Processing biomass

ABSTRACT

Biomass (e.g., plant biomass, animal biomass, microbial, and municipal waste biomass) is processed to produce useful products, such as food products and amino acids.

RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 14/750,995, filedJun. 25, 2015, which is a continuation of U.S. Ser. No. 14/333,675,filed Jul. 17, 2014, which is a continuation of U.S. Ser. No.13/902,246, filed May 24, 2013, now U.S. Pat. No. 8,877,472, issued onNov. 4, 2014, which is a continuation of U.S. Ser. No. 12/417,900, filedApr. 3, 2009, now abandoned, which claimed priority to U.S. ProvisionalApplication 61/139,453 filed Dec. 19, 2008, U.S. Provisional Application61/073,674 filed Jun. 18, 2008 and U.S. Provisional Application61/049,405 filed Apr. 30, 2008. The entirety of each of theseapplications is incorporated herein by reference.

TECHNICAL FIELD

This invention relates to processing biomass, to compositions includingsaccharide units arranged in a molecular chain, to methods of producingamino acids or antibiotics, to methods of producing edible orimmunostimulatory material, and to products of such methods.

BACKGROUND

Biomass, particularly biomass waste, is abundantly available. It wouldbe useful to derive products from biomass.

SUMMARY

Exemplary products that can be produced using the methods providedherein include foodstuffs suitable for use in, e.g., ingestion by ahuman and/or animal, aquaculture, agriculture, hydroponics,pharmaceuticals, nutraceuticals, pharmaceutical delivery vehicles anddosage forms, pharmaceutical excipients, pharmaceutical conjugates,cross-linked matrixes such as hydrogels, absorbent materials,fertilizers, and lignin products. Any product disclosed herein orproduced by the methods disclosed herein can be used as-is, or as aprecursor or an intermediate in the production of another product.

In many embodiments, products can be produced using Natural Force™Chemistry. Natural Force™ Chemistry methods use the controlledapplication and manipulation of physical forces, such as particle beams,gravity, light, etc., to create intended structural and chemicalmolecular change. In preferred implementations, Natural Force™ Chemistrymethods alter molecular structure without chemicals or microorganisms.By applying the processes of Nature, new useful matter can be createdwithout harmful environmental interference.

In one aspect, the present invention includes methods of preparing feedmaterials for animals (e.g., humans and animals, including but notlimited to food animals, pets, zoo animals, etc.), and for plants (e.g.,agricultural plants or crops or aquatic plants, in particular in ahydroponic solution or in aquaculture), and aquatic organisms (e.g.,fish, crustaceans, mollusks and the like).

These methods include obtaining a first material including biomass(e.g., plant biomass, animal biomass, microbial, and municipal wastebiomass) containing polysaccharides in the form of cellulose,hemicellulose, and/or starch. The molecular structure of thepolysaccharides of the first material is then modulated (e.g.,increased, decreased, or maintained) to produce a second material with agreater nutrient (e.g., protein, carbohydrate, fat, vitamin, and/ormineral) availability than the first material. The methods canoptionally include providing the second material to animals (e.g.,humans and/or non-human animals).

In some embodiments, the methods described herein can be used togenerate materials suitable for use in maintaining or promoting thegrowth of microorganisms (e.g., bacteria, yeast, fungi, protists, e.g.,an algae, protozoa or a fungus-like protist, e.g., a slime mold),aquatic organisms (e.g., in aquaculture), and/or plants and trees (e.g.,in agriculture, hydroponics and silvaculture).

In another aspect, the present invention provides methods of improvingthe pharmaceutical profile of materials. These methods include obtaininga first material including biomass (e.g., plant biomass, animal biomass,microbial, and municipal waste biomass) containing polysaccharides inthe form of cellulose, hemicellulose, and/or starch, and modulating(e.g., increasing, decreasing, or maintaining) the molecular structureof the polysaccharides of the first material to produce a secondmaterial, where one of the results of the methods is that thepharmaceutical profile of the second material is better or improved whencompared to the pharmaceutical profile of the first material. In someinstances, the methods include using first materials with little or nopharmaceutical profile prior to modulating the molecular structure ofthe first material. The second materials produced using the methodsdescribed herein are suitable for administration to an animal.

In a further aspect, the invention provides methods for obtaining aplant-derived pharmaceutical. These methods include processing amaterial including biomass (e.g., plant biomass, animal biomass,microbial, and municipal waste biomass) containing polysaccharides inthe form of cellulose, hemicellulose, and/or starch containing one ormore plant made pharmaceuticals, using any one or more of radiation,sonication, pyrolysis, and oxidation to obtain a plant-derivedpharmaceutical. In some instances the plant-derived made pharmaceuticalcan be isolated and/or purified.

In yet another aspect, the present invention provides methods ofpreparing nutraceuticals for human and/or a non-human animalconsumption. These methods include processing a material includingbiomass (e.g., plant biomass, animal biomass, microbial, and municipalwaste biomass) containing polysaccharides in the form of cellulose,hemicellulose, and/or starch so as to change the molecular structure ofthe polysaccharides of the material (e.g., increase or decrease themolecular weight of the material). These methods can optionally alsoinclude administering the resulting materials to humans and non-humananimals.

In an alternative aspect, the invention provides methods of preparingbiological agents and/or pharmaceutical agents. These method includeprocessing a material including biomass containing polysaccharides inthe form of cellulose, hemicellulose, and/or starch, so as to change themolecular structure of the polysaccharides of the material. Theresulting materials can then be combined with one or more biologicalagents and/or one or more pharmaceutical agents, which can beadministered to a subject.

Also provided in the present invention are methods of making hydrogels.These methods include processing a material including biomass containingpolysaccharides in the form of cellulose, hemicellulose, and/or starch,and changing the molecular structure of the polysaccharides to produce amaterial that includes cross-linked polymer chains. The method canfurther include cross-linking polymer chains in processed material.

In yet another aspect, the present invention provides methods of makingan absorbent or adsorbent material. These methods include processing amaterial including biomass containing polysaccharides in the form ofcellulose, hemicellulose, and/or starch, and changing the molecularstructure of the polysaccharides to produce an absorbent material. Theseabsorbent materials can be charged, e.g., positively or negativelycharged, and can have lipophilic and/or hydrophilic properties. As such,the materials can be used as animal litter or bedding, and/or absorbentmaterial to bind materials in a solution, (e.g., pollutants). In someembodiments, these absorbent materials can be used to bind biologicalmaterials in solutions of blood or plasma.

In a further aspect, the present invention provides methods of makingfertilizers. These methods include processing a material includingbiomass containing polysaccharides in the form of cellulose,hemicellulose, and/or starch, and changing the molecular structure ofthe polysaccharides to produce a material that has a greater solubilitythan the starting material and which is useful as a fertilizer.

Each of these methods includes treating the biomass using one or more of(e.g., one, two, three, or four of) size reduction (e.g., mechanicalsize reduction of individual pieces of biomass), radiation, sonication,pyrolysis, and oxidation to modulate the materials. In some embodiments,the methods use a radiation dose, e.g., from 0.1 Mrad to 10 Mrad. Insome embodiments, the methods use a radiation dose, e.g., from greaterthan 10 Mrad to 1000 Mrad.

In some aspects, the present invention also provides compositions madeusing any of the methods described herein. For example, the inventionfeatures a composition including saccharide units arranged in amolecular chain, wherein from about 1 out of ever 2 to about 1 out ofevery 250 saccharide units comprises a carboxylic acid group, or anester or a salt thereof, and the composition is suitable for consumptionas a feed material.

By “suitable for consumption as a feed material,” we mean that thecomposition is not toxic, under conditions of its intended use, to theliving being to which it is fed, and provides some nutritional value tothe being, e.g., energy and/or nutrients.

In some embodiments, the biomass feedstock is pretreated. In someembodiments, the methods disclosed herein can include a pre-treatment toreduce one or more dimensions of individual pieces of biomass. Forexample, pretreatment can include reducing one or more dimensions ofindividual pieces of biomass can include, e.g., shearing, cutting,crushing, smashing, or grinding.

Pressure can be utilized in all of the methods described herein. Forexample, at least one of the treating methods, e.g., radiation, can beperformed on the biomass under a pressure of greater than about 2.5atmospheres, such as greater than 5 or 10 atmospheres.

Examples of biomass (also referred to as ‘biomass feedstock’ or‘feedstock’) include cellulosic or lignocellulosic materials such aspaper, paper products, paper waste, wood, particle board, sawdust,agricultural waste, sewage, silage, grasses, rice hulls, bagasse,cotton, jute, hemp, flax, bamboo, sisal, abaca, straw, corn cobs, cornstover, switchgrass, alfalfa, hay, rice hulls, coconut hair, cotton,cassava, and synthetic celluloses and/or mixtures of these. In someinstances, biomass can include unicellular and/or multicellularorganisms. Exemplary organisms include, but are not limited to, e.g.,protists (e.g., animal (e.g., protozoa such as flagellates, amoeboids,ciliates, and sporozoa) and plant (e.g., algae such alveolates,chlorarachniophytes, cryptomonads, euglenids, glaucophytes, haptophytes,red algae, stramenopiles, and viridaeplantae)), seaweed, giant seaweed,water hyacinth, plankton (e.g., macroplankton, mesoplankton,microplankton, nanoplankton, picoplankton, and femptoplankton),phytoplankton, bacteria (e.g., gram positive bacteria, gram negativebacteria, and extremophiles), yeast and/or mixtures of these. In someinstances, biomass can include unicellular or multicellular organismsobtained from the ocean, lakes, and bodies of water including salt waterand fresh water. In some instances, biomass can include organic wastematerials such as animal waste or excrement or human waste or excrement(e.g., manure and sewage). In some instances, biomass can include anycombination of any of these. Other biomass materials are describedherein. Still other materials that include cellulose are described inthe patents, patent applications and publications that have beenincorporated by reference herein. In some instances, biomass can be,e.g., in solution, dry, and frozen.

If biomass is or includes microorganisms, these microorganisms willgenerally include carbohydrates, e.g., cellulose. These microorganismscan be in a solution, dry, frozen, active, and/or inactive state. Insome embodiments, these microorganisms can require additional processingprior to being subjected to the methods described herein. For example,the microorganisms can be in a solution and can be removed from thesolution, e.g., by centrifugation and/or filtration. Alternatively or inaddition, the microorganisms can be subjected to the methods describedherein without these additional steps, e.g., the microorganisms can beused in the solution. In some instances, the biomass can be or caninclude a natural or a synthetic material.

Irradiation can be, e.g., performed utilizing an ionizing radiation,such as gamma rays, a beam of electrons, or ultraviolet C radiationhaving a wavelength of from about 100 nm to about 280 nm. The ionizingradiation can include electron beam radiation. For example, theradiation can be applied at a total dose of between about 10 Mrad andabout 150 Mrad, such as at a dose rate of about 0.5 to about 10Mrad/day, or 1 Mrad/s to about 10 Mrad/s. In some embodiments,irradiating includes applying two or more radiation sources, such asgamma rays and a beam of electrons.

In some embodiments, the biomass exhibits a first level of recalcitranceand the carbohydrate material exhibits a second level of recalcitrancethat is lower that the first level of recalcitrance. For example, thesecond level of recalcitrance can be lower than the first level ofrecalcitrance by at least about 10% (e.g., 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 99%, 100%). In some embodiments, the level of recalcitrancecan be reduced by 50%-90%.

The biomass can be prepared by shearing biomass (e.g., a biomass fibersource) to provide a fibrous material. For example, the shearing can beperformed with a rotary knife cutter. The fibers of the fibrous materialcan have, e.g., an average length-to-diameter ratio (L/D) of greaterthan 5/1. The fibrous material can have, e.g., a BET surface area ofgreater than 0.25 m²/g (e.g., 0.3 m²/g, 0.35 m²/g, 0.35 m²/g, 0.4 m²/g,0.5 m²/g, 1 m²/g, 1.5 m²/g, 2 m²/g, 3 m²/g, 10 m²/g, 25 m²/g, or greaterthan 25 m²/g).

In some embodiments, the carbohydrate can include one or moreβ-1,4-linkages and have a number average molecular weight between about3,000 and 50,000 daltons.

In some examples, the pretreated biomass material can further include abuffer, such as sodium bicarbonate or ammonium chloride, an electrolyte,such as potassium chloride or sodium chloride a growth factor, such asbiotin and/or a base pair such as uracil, a surfactant, a mineral, or achelating agent.

To aid in the reduction of the molecular weight of the cellulose, anenzyme, e.g., a cellulolytic enzyme, and/or a swelling agent, can beutilized with any method described herein.

When a microorganism is utilized, it can be a natural microorganism oran engineered microorganism (e.g., a genetically modified microorganism(GMM)). For example, the microorganism can be a bacterium, e.g., acellulolytic bacterium, a fungus, e.g., a yeast, a plant or a protist,e.g., an algae, a protozoa or a fungus-like protist, e.g., a slime mold,protists (e.g., animal (e.g., protozoa such as flagellates, amoeboids,ciliates, and sporozoa) and plant (e.g., algae such alveolates,chlorarachniophytes, cryptomonads, euglenids, glaucophytes, haptophytes,red algae, stramenopiles, and viridaeplantae)), seaweed, plankton (e.g.,macroplankton, mesoplankton, microplankton, nanoplankton, picoplankton,and femptoplankton), phytoplankton, and/or mixtures of these. In someembodiments, the microorganism is white rot fungus. In some instances,the microorganism can include unicellular and/or multicellularorganisms, e.g., the ocean, lakes, and bodies of water including saltwater and fresh water. When the organisms are compatible, mixtures canbe utilized.

Generally, various microorganisms can produce a number of usefulproducts by operating on, converting, bioconverting, or fermenting thematerials. For example, alcohols, organic acids, hydrocarbons, hydrogen,proteins, carbohydrates, fats/oils/lipids, amino acids, vitamins, ormixtures of any of these materials can be produced by fermentation orother processes.

Examples of products that can be produced include mono- andpolyfunctional C1-C6 alkyl alcohols, mono- and poly-functionalcarboxylic acids, C1-C6 hydrocarbons, and combinations thereof. Specificexamples of suitable alcohols include methanol, ethanol, propanol,isopropanol, butanol, ethylene glycol, propylene glycol, 1,4-butanediol, glycerin, and combinations thereof. Specific example of suitablecarboxylic acids include formic acid, acetic acid, propionic acid,butyric acid, valeric acid, caproic acid, palmitic acid, stearic acid,oxalic acid, malonic acid, succinic acid, glutaric acid, oleic acid,linoleic acid, glycolic acid, lactic acid, γ-hydroxybutyric acid, andcombinations thereof. Examples of suitable hydrocarbons include methane,ethane, propane, pentane, n-hexane, and combinations thereof.

Another aspect of the invention features a method that includesconverting a low molecular weight sugar, or a material that includes alow molecular weight sugar, in a mixture with a biomass, amicroorganism, and a solvent or a solvent system, e.g., water or amixture of water and an organic solvent, to any product describedherein. Without wishing to be bound by any particular theory, it isbelieved that having a solid present, such as a high surface area and/orhigh porosity solid, can increase reaction rates by increasing theeffective concentration of solutes and providing a substrate on whichreactions can occur. Additional details about such a conversion aredescribed in U.S. patent application Ser. No. 12/417,840, filed Apr. 3,2009, the entire contents of which is hereby incorporated by referenceherein in its entirety.

The term “fibrous material,” as used herein, is a material that includesnumerous loose, discrete and separable fibers. For example, a fibrousmaterial can be prepared from a bleached Kraft paper fiber source byshearing, e.g., with a rotary knife cutter.

The term “screen,” as used herein, means a member capable of sievingmaterial according to size. Examples of screens include a perforatedplate, cylinder or the like, or a wire mesh or cloth fabric.

The term “pyrolysis,” as used herein, means to break bonds in a materialby the application of heat energy. Pyrolysis can occur while the subjectmaterial is under vacuum, or immersed in a gaseous material, such as anoxidizing gas, e.g., air or oxygen, or a reducing gas, such as hydrogen.

Oxygen content is measured by elemental analysis by pyrolyzing a samplein a furnace operating at 1300° C. or above.

For the purposes of this disclosure, carbohydrates are materials thatare composed entirely of one or more saccharide units or that includeone or more saccharide units. The saccharide units can be functionalizedabout the ring with one or more functional groups, such as carboxylicacid groups, amino groups, nitro groups, nitroso groups or nitrilegroups and still be considered carbohydrates. Carbohydrates can bepolymeric (e.g., equal to or greater than 10-mer, 100-mer, 1,000-mer,10,000-mer, or 100,000-mer), oligomeric (e.g., equal to or greater thana 4-mer, 5-mer, 6-mer, 7-mer, 8-mer, 9-mer or 10-mer), trimeric,dimeric, or monomeric. When the carbohydrates are formed of more than asingle repeat unit, each repeat unit can be the same or different.

Examples of polymeric carbohydrates include cellulose, xylan, pectin,and starch, while cellobiose and lactose are examples of dimericcarbohydrates. Examples of monomeric carbohydrates include glucose andxylose.

Carbohydrates can be part of a supramolecular structure, e.g.,covalently bonded into the structure. Examples of such materials includelignocellulosic materials, such as that found in wood.

A starchy material is one that is or includes significant amounts ofstarch or a starch derivative, such as greater than about 5 percent byweight starch or starch derivative. For purposes of this disclosure, astarch is a material that is or includes an amylose, an amylopectin, ora physical and/or chemical mixture thereof, e.g., a 20:80 or 30:70percent by weight mixture of amylose to amylopectin. For example, rice,corn, and mixtures thereof are starchy materials. Starch derivativesinclude, e.g., maltodextrin, acid-modified starch, base-modified starch,bleached starch, oxidized starch, acetylated starch, acetylated andoxidized starch, phosphate-modified starch, genetically-modified starchand starch that is resistant to digestion.

For purposes of this disclosure, a low molecular weight sugar is acarbohydrate or a derivative thereof that has a formula weight(excluding moisture) that is less than about 2,000, e.g., less thanabout 1,800, 1,600, less than about 1,000, less than about 500, lessthan about 350 or less than about 250. For example, the low molecularweight sugar can be a monosaccharide, e.g., glucose or xylose, adisaccharide, e.g., cellobiose or sucrose, or a trisaccharide.

Swelling agents as used herein are materials that cause a discernableswelling, e.g., a 2.5 percent increase in volume over an unswollen stateof biomass materials, when applied to such materials as a solution,e.g., a water solution. Examples include alkaline substances, such assodium hydroxide, potassium hydroxide, lithium hydroxide and ammoniumhydroxides, acidifying agents, such as mineral acids (e.g., sulfuricacid, hydrochloric acid and phosphoric acid), salts, such as zincchloride, calcium carbonate, sodium carbonate, benzyltrimethylammoniumsulfate, and basic organic amines, such as ethylene diamine.

In some embodiments of the methods described herein, no chemicals, e.g.,no swelling agents, are added to the biomass, e.g., none prior toirradiation. For example, alkaline substances (such as sodium hydroxide,potassium hydroxide, lithium hydroxide and ammonium hydroxides),acidifying agents (such as mineral acids (e.g., sulfuric acid,hydrochloric acid and phosphoric acid)), salts, such as zinc chloride,calcium carbonate, sodium carbonate, benzyltrimethylammonium sulfate, orbasic organic amines, such as ethylene diamine, is added prior toirradiation or other processing. In some cases, no additional water isadded. For example, the biomass prior to processing can have less than0.5 percent by weight added chemicals, e.g., less than 0.4, 0.25, 0.15,or 0.1 percent by weight added chemicals. In some instances, the biomasshas no more than a trace, e.g., less than 0.05 percent by weight addedchemicals, prior to irradiation. In other instances, the biomass priorto irradiation has substantially no added chemicals or swelling agents.Avoiding the use of such chemicals can also be extended throughout,e.g., at all times prior to fermentation, or at all times.

The term “edible,” as used herein, means fit to be eaten as food.

A “sheared material,” as used herein, is a material that includesdiscrete fibers in which at least about 50% of the discrete fibers, havea length/diameter (L/D) ratio of at least about 5, and that has anuncompressed bulk density of less than about 0.6 g/cm³.

In some embodiments, changing a molecular structure of biomass, as usedherein, means to change the chemical bonding arrangement, such as thetype and quantity of functional groups or conformation of the structure.For example, the change in the molecular structure can include changingthe recalcitrance level of the material, changing the supramolecularstructure of the material, oxidation of the material, changing anaverage molecular weight, changing an average crystallinity, changing asurface area, changing a degree of polymerization, changing a porosity,changing a degree of branching, grafting on other materials, changing acrystalline domain size, or an changing an overall domain size.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

As used herein, the term “subject” is used throughout the specificationto describe an animal, human, or non-human. The term includes, but isnot limited to, birds, reptiles, fish, plants, amphibians, and mammals,e.g., humans, other primates, pigs, rodents such as mice and rats,rabbits, guinea pigs, hamsters, cows, horses, cats, dogs, sheep andgoats.

The full disclosure of WO2008/073186 is incorporated by reference hereinin its entirety. The full disclosures of each of the following U.S.patents and published patent applications, which are being filedconcurrently herewith, are hereby incorporated by reference herein: U.S.Pat. No. 7,867,358 B2 (to Medoff), U.S. Pat. No. 7,846,295 B1 (toMedoff), U.S. Pat. No. 7,931,784 B2 (to Medoff), U.S. Pat. No. 8,236,535B2 (to Medoff and Masterman), U.S. Pat. App. Pub. No. 2010/0093241 A1(by Medoff), U.S. Pat. No. 8,212,087 B2 (to Medoff), U.S. Pat. App. Pub.No. 2009/0312537 A1 (by Medoff), U.S. Pat. No. 8,025,098 B2 (to Medoff),and U.S. Pat. No. 7,867,359 B2 (to Medoff).

Any carbohydrate material described herein can be utilized in anyapplication or process described in any patent or patent applicationincorporated by reference herein.

In any of the methods disclosed herein, radiation may be applied from adevice that is in a vault.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a block diagram illustrating conversion of biomass intoproducts and co-products.

FIG. 2 is block diagram illustrating conversion of a fiber source into afirst and second fibrous material.

FIG. 3 is a cross-sectional view of a rotary knife cutter.

FIG. 4 is block diagram illustrating conversion of a fiber source into afirst, second and third fibrous material.

FIG. 5 is block diagram illustrating densification of a material.

FIG. 6 is a perspective view of a pellet mill.

FIG. 7A is a densified fibrous material in pellet form.

FIG. 7B is a transverse cross-section of a hollow pellet in which acenter of the hollow is in-line with a center of the pellet.

FIG. 7C is a transverse cross-section of a hollow pellet in which acenter of the hollow is out of line with the center of the pellet.

FIG. 7D is a transverse cross-section of a tri-lobal pellet.

FIG. 8 is a block diagram illustrating a treatment sequence forprocessing feedstock.

FIG. 9 is a perspective, cut-away view of a gamma irradiator housed in aconcrete vault.

FIG. 10 is an enlarged perspective view of region R of FIG. 9.

FIG. 11 is a block diagram illustrating an electron beam irradiationfeedstock pretreatment sequence.

FIG. 11A is a schematic representation of biomass being ionized, andthen oxidized or quenched.

FIG. 11B is a schematic side view of a system for irradiating a low bulkdensity material, while FIG. 11C is cross-sectional of the system takenalong 11C-11C.

FIG. 11D is a schematic cross-sectional view of a fluidized bed systemfor irradiating a low bulk density material.

FIG. 11E is a schematic side-view of another system for irradiating alow bulk density material.

FIG. 12 is a schematic view of a system for sonicating a process streamof cellulosic material in a liquid medium.

FIG. 13 is a schematic view of a sonicator having two transducerscoupled to a single horn.

FIG. 14 is a block diagram illustrating a pyrolytic feedstockpretreatment system.

FIG. 15 is a cross-sectional side view of a pyrolysis chamber.

FIG. 16 is a cross-sectional side view of a pyrolysis chamber.

FIG. 17 is a cross-sectional side view of a pyrolyzer that includes aheated filament.

FIG. 18 is a schematic cross-sectional side view of a Curie-Pointpyrolyzer.

FIG. 19 is a schematic cross-sectional side view of a furnace pyrolyzer.

FIG. 20 is a schematic cross-sectional top view of a laser pyrolysisapparatus.

FIG. 21 is a schematic cross-sectional top view of a tungsten filamentflash pyrolyzer.

FIG. 22 is a block diagram illustrating an oxidative feedstockpretreatment system.

FIG. 23 is block diagram illustrating a general overview of the processof converting a fiber source into a product, e.g., ethanol.

FIG. 24 is a cross-sectional view of a steam explosion apparatus.

FIG. 25 is a schematic cross-sectional side view of a hybrid electronbeam/sonication device.

FIG. 26 is a scanning electron micrograph of a fibrous material producedfrom polycoated paper at 25× magnification. The fibrous material wasproduced on a rotary knife cutter utilizing a screen with ⅛ inchopenings.

FIG. 27 is a scanning electron micrograph of a fibrous material producedfrom bleached Kraft board paper at 25× magnification. The fibrousmaterial was produced on a rotary knife cutter utilizing a screen with ⅛inch openings.

FIG. 28 is a scanning electron micrograph of a fibrous material producedfrom bleached Kraft board paper at 25× magnification. The fibrousmaterial was twice sheared on a rotary knife cutter utilizing a screenwith 1/16 inch openings during each shearing.

FIG. 29 is a scanning electron micrograph of a fibrous material producedfrom bleached Kraft board paper at 25× magnification. The fibrousmaterial was thrice sheared on a rotary knife cutter. During the firstshearing, a ⅛ inch screen was used; during the second shearing, a 1/16inch screen was used, and during the third shearing a 1/32 inch screenwas used.

FIG. 30 is a schematic side view of a sonication apparatus, while FIG.31 is a cross-sectional view through the processing cell of FIG. 30.

FIG. 32 is a scanning electron micrograph at 1000× magnification of afibrous material produced from shearing switchgrass on a rotary knifecutter, and then passing the sheared material through a 1/32 inchscreen.

FIGS. 33 and 34 are scanning electron micrographs of the fibrousmaterial of FIG. 32 after irradiation with 10 Mrad and 100 Mrad gammarays, respectively, at 1000× magnification.

FIG. 35 is a scanning electron micrographs of the fibrous material ofFIG. 32 after irradiation with 10 Mrad and sonication at 1000×magnification.

FIG. 36 is a scanning electron micrographs of the fibrous material ofFIG. 32 after irradiation with 100 Mrad and sonication at 1000×magnification.

FIG. 37 is an infrared spectrum of Kraft board paper sheared on a rotaryknife cutter.

FIG. 38 is an infrared spectrum of the Kraft paper of FIG. 37 afterirradiation with 100 Mrad of gamma radiation.

FIG. 39 is a schematic view of a process for biomass conversion.

FIG. 40 is schematic view of another process for biomass conversion.

FIG. 41 is a schematic diagram of a truck-based mobile biomassprocessing facility.

FIG. 42 is a schematic diagram of a train-based mobile biomassprocessing facility.

FIGS. 43A and 43B are schematic diagrams showing the processing stepsfor generating products and co-products from biomass (A) and forgenerating products using a bioconversion step.

FIG. 44 is a schematic diagram showing a variable volume fed-batchfermentation process.

FIG. 45 is a schematic diagram showing a fixed volume fed-batchfermentation process.

FIG. 46 is a schematic diagram showing the processing steps required forthe production of products 1, 2, and 3. Star indicates a step isoptional. Black arrow indicates that an optional densification step canbe performed.

FIG. 47 is a diagram illustrating the biosynthesis of aromatic aminoacids such as tryptophan, phenylalanine, and tyrosine through theshikimic acid pathway.

FIGS. 48A through 48F are diagrams illustrating antibiotics and othernatural products that can be generated by microorganisms.

FIG. 49 is a diagram illustrating the biosynthesis of streptomycin,starting from D-glucose.

FIG. 50 is a diagram illustrating the biosynthesis of vancomycin,starting from a glucose derivative.

FIG. 51 is a diagram illustrating the biosynthesis of ansamycin,starting from a glucose derivative.

DETAILED DESCRIPTION

Biomass (e.g., plant biomass, animal biomass, microbial biomass, andmunicipal waste biomass) can be processed using the methods disclosedherein to produce useful products such as food products. In addition,functionalized materials having desired types and amounts offunctionality, such as carboxylic acid groups, aldehyde groups, ketonegroups, nitrile groups, nitro groups, or nitroso groups, can be preparedusing the methods described herein. Such functionalized materials canbe, e.g., more soluble, easier to utilize by various microorganisms orcan be more stable over the long term, e.g., less prone to oxidation.Systems and processes are described below herein that can use variousbiomass materials, e.g., cellulosic materials, lignocellulosicmaterials, starchy materials, or materials that are or that include lowmolecular weight sugars, as feedstock materials. Biomass materials areoften readily available, can be difficult to process, e.g., byfermentation, or can give sub-optimal yields at a slow rate, forexample, by fermentation. Biomass materials can be first pretreated,often by size reduction of raw feedstock materials. Pretreated biomasscan then be treated using at least one of: radiation (under controlledthermal conditions), sonication, oxidation, pyrolysis, and steamexplosion. The various pretreatment systems and methods can be used incombinations of two, three, or even four of these technologies.

Alternatively or in addition, the present invention is based, at leastin part, on the observation that the methods described herein can beused to convert biomass into non-energy materials and compositions. Suchmaterials and compositions include, but are not limited to, foodstuffs(e.g., suitable for consumption by humans and/or animals),pharmaceuticals, nutraceuticals, pharmaceutical delivery vehicles anddosage forms, pharmaceutical excipients, pharmaceutical conjugates,cross-linked matrixes such as hydrogels, absorbent materials,fertilizers, and lignin products.

Types of Biomass

Generally, any biomass material that is or includes carbohydratescomposed entirely of one or more saccharide units or that include one ormore saccharide units can be processed by any of the methods describedherein. As used herein, biomass includes, cellulosic, lignocellulosic,hemicellulosic, starch, and lignin-containing materials. For example,the biomass material can be cellulosic or lignocellulosic materials, orstarchy materials, such as kernels of corn, grains of rice or otherfoods, or materials that are or that include one or more low molecularweight sugars, such as sucrose or cellobiose.

For example, such materials can include paper, paper products, wood,wood-related materials, particle board, grasses, rice hulls, bagasse,cotton, jute, hemp, flax, bamboo, sisal, abaca, straw, corn cobs, ricehulls, coconut hair, algae, seaweed (e.g., giant seaweed), waterhyacinth, cassava, coffee beans, coffee bean grounds (used coffee beangrounds), cotton, synthetic celluloses, or mixtures of any of these.

Fiber sources include cellulosic fiber sources, including paper andpaper products (e.g., polycoated paper and Kraft paper), andlignocellulosic fiber sources, including wood, and wood-relatedmaterials, e.g., particle board. Other suitable fiber sources includenatural fiber sources, e.g., grasses, rice hulls, bagasse, cotton, jute,hemp, flax, bamboo, sisal, abaca, straw, corn cobs, rice hulls, coconuthair; fiber sources high in α-cellulose content, e.g., cotton; andsynthetic fiber sources, e.g., extruded yarn (oriented yarn orun-oriented yarn). Natural or synthetic fiber sources can be obtainedfrom virgin scrap textile materials, e.g., remnants or they can be postconsumer waste, e.g., rags. When paper products are used as fibersources, they can be virgin materials, e.g., scrap virgin materials, orthey can be post-consumer waste. Aside from virgin raw materials,post-consumer, industrial (e.g., offal), and processing waste (e.g.,effluent from paper processing) can also be used as fiber sources. Also,the fiber source can be obtained or derived from human (e.g., sewage),animal, or plant waste. Additional fiber sources have been described inthe art, for example, see U.S. Pat. Nos. 6,448,307, 6,258,876,6,207,729, 5,973,035 and 5,952,105.

Microbial sources include, but are not limited to, any naturallyoccurring or genetically modified microorganism or organism thatcontains or are capable of providing a source of carbohydrates (e.g.,cellulose), for example, protists (e.g., animal (e.g., protozoa such asflagellates, amoeboids, ciliates, and sporozoa) and plant (e.g., algaesuch alveolates, chlorarachniophytes, cryptomonads, euglenids,glaucophytes, haptophytes, red algae, stramenopiles, andviridaeplantae)), seaweed, plankton (e.g., macroplankton, mesoplankton,microplankton, nanoplankton, picoplankton, and femptoplankton),phytoplankton, bacteria (e.g., gram positive bacteria, gram negativebacteria, and extremophiles), yeast and/or mixtures of these. In someinstances, microbial biomass can be obtained from natural sources, e.g.,the ocean, lakes, bodies of water, e.g., salt water or fresh water, oron land. Alternatively or in addition, microbial biomass can be obtainedfrom culture systems, e.g., large scale dry and wet culture systems.

Examples of biomass include renewable, organic matter, such as plantbiomass, microbial biomass, animal biomass (e.g., any animal by-product,animal waste, etc.) and municipal waste biomass including any and allcombinations of these biomass materials.

Plant biomass and lignocellulosic biomass include organic matter (woodyor non-woody) derived from plants, especially matter available on asustainable basis. Examples include biomass from agricultural or foodcrops (e.g., sugarcane, sugar beets or corn kernels) or an extracttherefrom (e.g., sugar from sugarcane and corn starch from corn),agricultural crop wastes and residues such as corn stover, wheat straw,rice straw, sugar cane bagasse, and the like. Plant biomass furtherincludes, but is not limited to, trees, woody energy crops, wood wastesand residues such as softwood forest thinnings, barky wastes, sawdust,paper and pulp industry waste streams, wood fiber, and the like.Additionally grass crops, such as switchgrass and the like havepotential to be produced on a large-scale as another plant biomasssource. For urban areas, the plant biomass feedstock includes yard waste(e.g., grass clippings, leaves, tree clippings, and brush) and vegetableprocessing waste.

In some embodiments, biomass can include lignocellulosic feedstock canbe plant biomass such as, but not limited to, non-woody plant biomass,cultivated crops, such as, but not limited to, grasses, for example, butnot limited to, C4 grasses, such as switchgrass, cord grass, rye grass,miscanthus, reed canary grass, or a combination thereof, or sugarprocessing residues such as bagasse, or beet pulp, agriculturalresidues, for example, soybean stover, corn stover, rice straw, ricehulls, barley straw, corn cobs, wheat straw, canola straw, rice straw,oat straw, oat hulls, corn fiber, recycled wood pulp fiber, sawdust,hardwood, for example aspen wood and sawdust, softwood, or a combinationthereof. Further, the lignocellulosic feedstock can include cellulosicwaste material such as, but not limited to, newsprint, cardboard,sawdust, and the like. Lignocellulosic feedstock can include one speciesof fiber or alternatively, lignocellulosic feedstock can include amixture of fibers that originate from different lignocellulosicfeedstocks. Furthermore, the lignocellulosic feedstock can comprisefresh lignocellulosic feedstock, partially dried lignocellulosicfeedstock, fully dried lignocellulosic feedstock, or a combinationthereof.

Microbial biomass includes biomass derived from naturally occurring orgenetically modified unicellular organisms and/or multicellularorganisms, e.g., organisms from the ocean, lakes, bodies of water, e.g.,salt water or fresh water, or on land, and that contains a source ofcarbohydrate (e.g., cellulose). Microbial biomass can include, but isnot limited to, for example protists (e.g., animal (e.g., protozoa suchas flagellates, amoeboids, ciliates, and sporozoa) and plant (e.g.,algae such alveolates, chlorarachniophytes, cryptomonads, euglenids,glaucophytes, haptophytes, red algae, stramenopiles, andviridaeplantae)), seaweed, plankton (e.g., macroplankton, mesoplankton,microplankton, nanoplankton, picoplankton, and femptoplankton),phytoplankton, bacteria (e.g., gram positive bacteria, gram negativebacteria, and extremophiles), yeast and/or mixtures of these. In someinstances, microbial biomass can be obtained from natural sources, e.g.,the ocean, lakes, bodies of water, e.g., salt water or fresh water, oron land. Alternatively or in addition, microbial biomass can be obtainedfrom culture systems, e.g., large scale dry and wet culture systems.

Animal biomass includes any organic waste material such asanimal-derived waste material or excrement or human waste material orexcrement (e.g., manure and sewage).

In some embodiments, the carbohydrate is or includes a material havingone or more β-1,4-linkages and having a number average molecular weightbetween about 3,000 and 50,000. Such a carbohydrate is or includescellulose (I), which is derived from (β-glucose 1) through condensationof β(1→4)-glycosidic bonds. This linkage contrasts itself with that forα(1→4)-glycosidic bonds present in starch and other carbohydrates.

Starchy materials include starch itself, e.g., corn starch, wheatstarch, potato starch or rice starch, a derivative of starch, or amaterial that includes starch, such as an edible food product or a crop.For example, the starchy material can be arracacha, buckwheat, banana,barley, cassava, kudzu, oca, sago, sorghum, regular household potatoes,sweet potato, taro, yams, or one or more beans, such as favas, lentilsor peas. Blends of any one or more starchy material is also a starchymaterial. In particular embodiments, the starchy material is derivedfrom corn. Various corn starches and derivatives are known in the art,see, e.g., “Corn Starch,” Corn Refiners Association (11th Edition,2006).

Biomass materials that include low molecular weight sugars can, e.g.,include at least about 0.5 percent by weight of the low molecular sugar,e.g., at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12.5, 25, 35, 50, 60,70, 80, 90 or even at least about 95 percent by weight of the lowmolecular weight sugar. In some instances, the biomass is composedsubstantially of the low molecular weight sugar, e.g., greater than 95percent by weight, such as 96, 97, 98, 99 or substantially 100 percentby weight of the low molecular weight sugar.

Biomass materials that include low molecular weight sugars can beagricultural products or food products, such as sugarcane and sugarbeets or an extract therefrom, e.g., juice from sugarcane, or juice fromsugar beets. Biomass materials that include low molecular weight sugarscan be substantially pure extracts, such as raw or crystallized tablesugar (sucrose). Low molecular weight sugars include sugar derivatives.For example, the low molecular weight sugars can be oligomeric (e.g.,equal to or greater than a 4-mer, 5-mer, 6-mer, 7-mer, 8-mer, 9-mer or10-mer), trimeric, dimeric, or monomeric. When the carbohydrates areformed of more than a single repeat unit, each repeat unit can be thesame or different.

Specific examples of low molecular weight sugars include cellobiose,lactose, sucrose, glucose and xylose, along with derivatives thereof. Insome instances, sugar derivatives are more rapidly dissolved in solutionor utilized by microbes to provide a useful material. Several suchsugars and sugar derivatives are shown below.

Combinations (e.g., by themselves or in combination of any biomassmaterial, component, product, and/or co-product generated using themethods described herein) of any biomass materials described herein canbe utilized for making any of the products described herein. Forexample, blends of cellulosic materials and starchy materials can beutilized for making any product described herein.

Systems for Treating Biomass

FIG. 1 shows a system 100 for converting biomass, particularly biomasswith significant cellulosic and lignocellulosic components and/orstarchy components, into useful products and co-products. System 100includes a feed preparation subsystem 110, a pretreatment subsystem 114,a primary process subsystem 118, and a post-processing subsystem 122.Feed preparation subsystem 110 receives biomass in its raw form,physically prepares the biomass for use as feedstock by downstreamprocesses (e.g., reduces the size of and homogenizes the biomass), andstores the biomass both in its raw and feedstock forms.

Biomass feedstock with significant cellulosic and/or lignocellulosiccomponents, or starchy components can have a high average molecularweight and crystallinity that can make processing the feedstock intouseful products (e.g., fermenting the feedstock to produce ethanol)difficult. Accordingly it is useful to treat biomass feedstock, e.g.,using the treatment methods described herein. As described herein, insome embodiments, the treatment of biomass does not use acids, basesand/or enzymes to process biomass, or only uses such treatments in smallor catalytic amounts.

Treatment subsystem 114 receives biomass feedstock from the feedpreparation subsystem 110 and prepares the feedstock for use in primaryproduction processes by, for example, reducing the average molecularweight and crystallinity of the feedstock. Primary process subsystem 118receives treated feedstock from treatment subsystem 114 and producesuseful products (e.g., ethanol, other alcohols, pharmaceuticals, and/orfood products). In some cases, the output of primary process subsystem118 is directly useful but, in other cases, requires further processingprovided by post-processing subsystem 122. Post-processing subsystem 122provides further processing to product streams from primary processsystem 118 which require it (e.g., distillation and denaturation ofethanol) as well as treatment for waste streams from the othersubsystems. In some cases, the co-products of subsystems 114, 118, 122can also be directly or indirectly useful as secondary products and/orin increasing the overall efficiency of system 100. For example,post-processing subsystem 122 can produce treated water to be recycledfor use as process water in other subsystems and/or can produce burnablewaste which can be used as fuel for boilers producing steam and/orelectricity.

The optimum size for biomass conversion plants is affected by factorsincluding economies of scale and the type and availability of biomassused as feedstock. Increasing plant size tends to increase economies ofscale associated with plant processes. However, increasing plant sizealso tends to increase the costs (e.g., transportation costs) per unitof feedstock. Studies analyzing these factors suggest that theappropriate size for biomass conversion plants can range from 100 to1,000 or more, e.g., 10,000 or more dried tons of feedstock per daydepending at least in part on the type of feedstock used. The type ofbiomass feedstock can also impact plant storage requirements with plantsdesigned primarily for processing feedstock whose availability variesseasonally (e.g., corn stover) requiring more on- or off-site feedstockstorage than plants designed to process feedstock whose availability isrelatively steady (e.g., waste paper).

Biomass Pretreatment

In some cases, pretreatment methods of processing begin with a physicalpreparation of the biomass, e.g., size reduction of raw biomassfeedstock materials, such as by cutting, grinding, crushing, smashing,shearing or chopping. In some embodiments, methods (e.g., mechanicalmethods) are used to reduce the size and/or dimensions of individualpieces of biomass. In some cases, loose feedstock (e.g., recycled paperor switchgrass) is pretreated by shearing or shredding. Screens and/ormagnets can be used to remove oversized or undesirable objects such as,for example, rocks or nails from the feed stream.

Feed pretreatment systems can be configured to produce feed streams withspecific characteristics such as, for example, specific maximum sizes,specific length-to-width, or specific surface areas ratios. As a part offeed pretreatment, the bulk density of feedstocks can be controlled(e.g., increased).

Size Reduction

In some embodiments, the biomass is in the form of a fibrous materialthat includes fibers provided by shearing the biomass. For example, theshearing can be performed with a rotary knife cutter.

For example, and by reference to FIG. 2, a biomass fiber source 210 issheared, e.g., in a rotary knife cutter, to provide a first fibrousmaterial 212. The first fibrous material 212 is passed through a firstscreen 214 having an average opening size of 1.59 mm or less ( 1/16inch, 0.0625 inch) to provide a second fibrous material 216. If desired,fiber source can be cut prior to the shearing, e.g., with a shredder.For example, when a paper is used as the fiber source, the paper can befirst cut into strips that are, e.g., ¼- to ½-inch wide, using ashredder, e.g., a counter-rotating screw shredder, such as thosemanufactured by Munson (Utica, N.Y.). As an alternative to shredding,the paper can be reduced in size by cutting to a desired size using aguillotine cutter. For example, the guillotine cutter can be used to cutthe paper into sheets that are, e.g., 10 inches wide by 12 inches long.

In some embodiments, the shearing of fiber source and the passing of theresulting first fibrous material through first screen are performedconcurrently. The shearing and the passing can also be performed in abatch-type process.

For example, a rotary knife cutter can be used to concurrently shear thefiber source and screen the first fibrous material. Referring to FIG. 3,a rotary knife cutter 220 includes a hopper 222 that can be loaded witha shredded fiber source 224 prepared by standard methods. Shredded fibersource is sheared between stationary blades 230 and rotating blades 232to provide a first fibrous material 240. First fibrous material 240passes through screen 242, and the resulting second fibrous material 244is captured in bin 250. To aid in the collection of the second fibrousmaterial, the bin can have a pressure below nominal atmosphericpressure, e.g., at least 10 percent below nominal atmospheric pressure,e.g., at least 25 percent below nominal atmospheric pressure, at least50 percent below nominal atmospheric pressure, or at least 75 percentbelow nominal atmospheric pressure. In some embodiments, a vacuum source252 is utilized to maintain the bin below nominal atmospheric pressure.

Shearing can be advantageous for “opening up” and “stressing” thefibrous materials, making the cellulose of the materials moresusceptible to chain scission and/or reduction of crystallinity. Theopen materials can also be more susceptible to oxidation whenirradiated.

In some embodiments, shearing can be advantageous for “opening up” and“stressing” the fibrous materials, making the cellulose of the materialsmore susceptible to ruminant digestion and absorption.

The fiber source can be sheared in a dry state, a hydrated state (e.g.,having up to ten percent by weight absorbed water), or in a wet state,e.g., having between about 10 percent and about 75 percent by weightwater. The fiber source can even be sheared while partially or fullysubmerged under a liquid, such as water, ethanol, or isopropanol.

The fiber source can also be sheared in under a gas (such as a stream oratmosphere of gas other than air), e.g., oxygen or nitrogen, or insteam.

Other methods of making the fibrous materials include, e.g., stonegrinding, mechanical ripping or tearing, pin grinding, and/or airattrition milling.

If desired, the fibrous materials can be separated, e.g., continuouslyor in batches, into fractions according to their length, width, density,material type, or some combination of these attributes.

For example, ferrous materials can be separated from any of the fibrousmaterials by passing a fibrous material that includes a ferrous materialpast a magnet, e.g., an electromagnet, and then passing the resultingfibrous material through a series of screens, each screen havingdifferent sized apertures.

The fibrous materials can also be separated, e.g., by using a highvelocity gas, e.g., air. In such an approach, the fibrous materials areseparated by drawing off different fractions, which can be characterizedphotonically, if desired. Such a separation apparatus is discussed inLindsey et al, U.S. Pat. No. 6,883,667.

The fibrous materials can be pre-treated immediately following theirpreparation, or they can be dried, e.g., at approximately 105° C. for4-18 hours, so that the moisture content is, e.g., less than about 0.5%before use.

If desired, lignin can be removed from any of the fibrous materials thatinclude lignin. Also, to aid in the breakdown of the materials thatinclude the cellulose, the material can be treated prior to irradiationwith heat, a chemical (e.g., mineral acid, base or a strong oxidizersuch as sodium hypochlorite) and/or an enzyme.

In some embodiments, the average opening size of the first screen isless than 0.79 mm ( 1/32 inch, 0.03125 inch), e.g., less than 0.51 mm (1/50 inch, 0.02000 inch), less than 0.40 mm ( 1/64 inch, 0.015625 inch),less than 0.23 mm (0.009 inch), less than 0.20 mm ( 1/128 inch,0.0078125 inch), less than 0.18 mm (0.007 inch), less than 0.13 mm(0.005 inch), or even less than less than 0.10 mm ( 1/256 inch,0.00390625 inch). The screen is prepared by interweaving monofilamentshaving an appropriate diameter to give the desired opening size. Forexample, the monofilaments can be made of a metal, e.g., stainlesssteel. As the opening sizes get smaller, structural demands on themonofilaments can become greater. For example, for opening sizes lessthan 0.40 mm, it can be advantageous to make the screens frommonofilaments made from a material other than stainless steel, e.g.,titanium, titanium alloys, amorphous metals, nickel, tungsten, rhodium,rhenium, ceramics, or glass. In some embodiments, the screen is madefrom a plate, e.g., a metal plate, having apertures, e.g., cut into theplate using a laser. In some embodiments, the open area of the mesh isless than 52%, e.g., less than 41%, less than 36%, less than 31%, lessthan 30%.

In some embodiments, the second fibrous is sheared and passed throughthe first screen, or a different sized screen. In some embodiments, thesecond fibrous material is passed through a second screen having anaverage opening size equal to or less than that of first screen.

Referring to FIG. 4, a third fibrous material 220 can be prepared fromthe second fibrous material 216 by shearing the second fibrous material216 and passing the resulting material through a second screen 222having an average opening size less than the first screen 214.

Generally, the fibers of the fibrous materials can have a relativelylarge average length-to-diameter ratio (e.g., greater than 20-to-1),even if they have been sheared more than once. In addition, the fibersof the fibrous materials described herein can have a relatively narrowlength and/or length-to-diameter ratio distribution.

As used herein, average fiber widths (e.g., diameters) are thosedetermined optically by randomly selecting approximately 5,000 fibers.Average fiber lengths are corrected length-weighted lengths. BET(Brunauer, Emmet and Teller) surface areas are multi-point surfaceareas, and porosities are those determined by mercury porosimetry.

The average length-to-diameter ratio of the second fibrous material 14can be greater than 5/1, e.g., greater than 8/1, e.g., greater than10/1, greater than 15/1, greater than 20/1, greater than 25/1, orgreater than 50/1. An average length of the second fibrous material 14can be, e.g., between about 0.5 mm and 2.5 mm, e.g., between about 0.75mm and 1.0 mm, and an average width (e.g., diameter) of the secondfibrous material 14 can be, e.g., between about 5 μm and 50 μm, e.g.,between about 10 μm and 30 μm.

In some embodiments, a standard deviation of the length of the secondfibrous material 14 is less than 60 percent of an average length of thesecond fibrous material 14, e.g., less than 50 percent of the averagelength, less than 40 percent of the average length, less than 25 percentof the average length, less than 10 percent of the average length, lessthan 5 percent of the average length, or even less than 1 percent of theaverage length.

In some embodiments, a BET surface area of the second fibrous materialis greater than 0.1 m²/g, e.g., greater than 0.25 m²/g, greater than 0.5m²/g, greater than 1.0 m²/g, greater than 1.5 m²/g, greater than 1.75m²/g, greater than 5.0 m²/g, greater than 10 m²/g, greater than 25 m²/g,greater than 35 m²/g, greater than 50 m²/g, greater than 60 m²/g,greater than 75 m²/g, greater than 100 m²/g, greater than 150 m²/g,greater than 200 m²/g, or even greater than 250 m²/g. A porosity of thesecond fibrous material 14 can be, e.g., greater than 20 percent,greater than 25 percent, greater than 35 percent, greater than 50percent, greater than 60 percent, greater than 70 percent, e.g., greaterthan 80 percent, greater than 85 percent, greater than 90 percent,greater than 92 percent, greater than 94 percent, greater than 95percent, greater than 97.5 percent, greater than 99 percent, or evengreater than 99.5 percent.

In some embodiments, a ratio of the average length-to-diameter ratio ofthe first fibrous material to the average length-to-diameter ratio ofthe second fibrous material is, e.g., less than 1.5, e.g., less than1.4, less than 1.25, less than 1.1, less than 1.075, less than 1.05,less than 1.025, or even substantially equal to 1.

In particular embodiments, the second fibrous material is sheared againand the resulting fibrous material passed through a second screen havingan average opening size less than the first screen to provide a thirdfibrous material. In such instances, a ratio of the averagelength-to-diameter ratio of the second fibrous material to the averagelength-to-diameter ratio of the third fibrous material can be, e.g.,less than 1.5, e.g., less than 1.4, less than 1.25, or even less than1.1.

In some embodiments, the third fibrous material is passed through athird screen to produce a fourth fibrous material. The fourth fibrousmaterial can be, e.g., passed through a fourth screen to produce a fifthmaterial. Similar screening processes can be repeated as many times asdesired to produce the desired fibrous material having the desiredproperties.

Densification

As used herein, densification refers to increasing the bulk density of amaterial. Densified materials can be processed, or any processedmaterials can be densified, by any of the methods described herein.

A material, e.g., a fibrous material, having a low bulk density can bedensified to a product having a higher bulk density. For example, amaterial composition having a bulk density of 0.05 g/cm³ can bedensified by sealing the fibrous material in a relatively gasimpermeable structure, e.g., a bag made of polyethylene or a bag made ofalternating layers of polyethylene and a nylon, and then evacuating theentrapped gas, e.g., air, from the structure. After evacuation of theair from the structure, the fibrous material can have, e.g., a bulkdensity of greater than 0.3 g/cm³, e.g., 0.5 g/cm³, 0.6 g/cm³, 0.7 g/cm³or more, e.g., 0.85 g/cm³. After densification, the product canpre-treated by any of the methods described herein, e.g., irradiated,e.g., with gamma radiation. This can be advantageous when it isdesirable to transport the material to another location, e.g., a remotemanufacturing plant, where the fibrous material composition can be addedto a solution, e.g., to produce ethanol. After piercing thesubstantially gas impermeable structure, the densified fibrous materialcan revert to nearly its initial bulk density, e.g., to at least 60percent of its initial bulk density, e.g., 70 percent, 80 percent, 85percent or more, e.g., 95 percent of its initial bulk density. To reducestatic electricity in the fibrous material, an anti-static agent can beadded to the material.

In some embodiments, the structure, e.g., a carrier such as a bag, isformed of a material that dissolves in a liquid, such as water. Forexample, the structure can be formed from a polyvinyl alcohol so that itdissolves when in contact with a water-based solution. Such embodimentsallow densified structures to be added directly to solutions thatinclude a microorganism, without first releasing the contents of thestructure, e.g., by cutting.

Referring to FIG. 5, a biomass material can be combined with any desiredadditives and a binder, and subsequently densified by application ofpressure, e.g., by passing the material through a nip defined betweencounter-rotating pressure rolls or by passing the material through apellet mill. During the application of pressure, heat can optionally beapplied to aid in the densification of the fibrous material. Thedensified material can then be irradiated.

In some embodiments, the material prior to densification has a bulkdensity of less than 0.25 g/cm³, e.g., less than or about 0.20 g/cm³,0.15 g/cm³, 0.10 g/cm³, 0.05 g/cm³ or less, e.g., 0.025 g/cm³. Bulkdensity is determined using ASTM D1895B. Briefly, the method involvesfilling a measuring cylinder of known volume with a sample and obtaininga weight of the sample. The bulk density is calculated by dividing theweight of the sample in grams by the known volume of the cylinder incubic centimeters.

The preferred binders include binders that are soluble in water, swollenby water, or that have a glass transition temperature of less 25° C., asdetermined by differential scanning calorimetry. Water-soluble bindershave a solubility of at least about 0.05 weight percent in water. Waterswellable binders are binders that increase in volume by more than 0.5percent upon exposure to water.

In some embodiments, the binders that are soluble or swollen by waterinclude a functional group that is capable of forming a bond, e.g., ahydrogen bond, with the fibers of the fibrous material, e.g., cellulosicfibrous material. For example, the functional group can be a carboxylicacid group, a carboxylate group, a carbonyl group, e.g., of an aldehydeor a ketone, a sulfonic acid group, a sulfonate group, a phosphoric acidgroup, a phosphate group, an amide group, an amine group, a hydroxylgroup, e.g., of an alcohol, and combinations of these groups, e.g., acarboxylic acid group and a hydroxyl group. Specific monomeric examplesinclude glycerin, glyoxal, ascorbic acid, urea, glycine,pentaerythritol, a monosaccharide or a disaccharide, citric acid, andtartaric acid. Suitable saccharides include glucose, sucrose, lactose,ribose, fructose, mannose, arabinose and erythrose. Polymeric examplesinclude polyglycols, polyethylene oxide, polycarboxylic acids,polyamides, polyamines and polysulfonic acids polysulfonates. Specificpolymeric examples include polypropylene glycol (PPG), polyethyleneglycol (PEG), polyethylene oxide, e.g., POLYOX®, copolymers of ethyleneoxide and propylene oxide, polyacrylic acid (PAA), polyacrylamide,polypeptides, polyethylenimine, polyvinylpyridine,poly(sodium-4-styrenesulfonate) andpoly(2-acrylamido-methyl-1-propanesulfonic acid).

In some embodiments, the binder includes a polymer that has a glasstransition temperature less 25° C. Examples of such polymers includethermoplastic elastomers (TPEs). Examples of TPEs include polyetherblock amides, such as those available under the tradename PEBAX®,polyester elastomers, such as those available under the tradenameHYTREL®, and styrenic block copolymers, such as those available underthe tradename KRATON®. Other suitable polymers having a glass transitiontemperature less 25° C. include ethylene vinyl acetate copolymer (EVA),polyolefins, e.g., polyethylene, polypropylene, ethylene-propylenecopolymers, and copolymers of ethylene and alpha olefins, e.g.,1-octene, such as those available under the tradename ENGAGE®. In someembodiments, e.g., when the material is a fiberized polycoated paper,the material is densified without the addition of a separate low glasstransition temperature polymer.

In a particular embodiment, the binder is a lignin, e.g., a natural orsynthetically modified lignin.

A suitable amount of binder added to the material, calculated on a dryweight basis, is, e.g., from about 0.01 percent to about 50 percent,e.g., 0.03 percent, 0.05 percent, 0.1 percent, 0.25 percent, 0.5percent, 1.0 percent, 5 percent, 10 percent or more, e.g., 25 percent,based on a total weight of the densified material. The binder can beadded to the material as a neat, pure liquid, as a liquid having thebinder dissolved therein, as a dry powder of the binder, or as pelletsof the binder.

The densified fibrous material can be made in a pellet mill. Referringto FIG. 6, a pellet mill 300 has a hopper 301 for holding undensifiedmaterial 310 that includes carbohydrate-containing materials, such ascellulose. The hopper communicates with an auger 312 that is driven byvariable speed motor 314 so that undensified material can be transportedto a conditioner 320 that stirs the undensified material with paddles322 that are rotated by conditioner motor 330. Other ingredients, e.g.,any of the additives and/or fillers described herein, can be added atinlet 332. If desired, heat can be added while the fibrous material isin conditioner. After conditioned, the material passes from theconditioner through a dump chute 340, and to another auger 342. The dumpchute, as controlled by actuator 344, allows for unobstructed passage ofthe material from conditioner to auger. Auger is rotated by motor 346,and controls the feeding of the fibrous material into die and rollerassembly 350. Specifically, the material is introduced into a hollow,cylindrical die 352, which rotates about a horizontal axis and which hasradially extending die holes 250. Die 352 is rotated about the axis bymotor 360, which includes a horsepower gauge, indicating total powerconsumed by the motor. Densified material 370, e.g., in the form ofpellets, drops from chute 372 and are captured and processed, such as byirradiation.

The material, after densification, can be conveniently in the form ofpellets or chips having a variety of shapes. The pellets can then beirradiated. In some embodiments, the pellets or chips are cylindrical inshape, e.g., having a maximum transverse dimension of, e.g., 1 mm ormore, e.g., 2 mm, 3 mm, 5 mm, 8 mm, 10 mm, 15 mm or more, e.g., 25 mm.Other convenient shapes include pellets or chips that are plate-like inform, e.g., having a thickness of 1 mm or more, e.g., 2 mm, 3 mm, 5 mm,8 mm, 10 mm or more, e.g., 25 mm; a width of, e.g., 5 mm or more, e.g.,10 mm, 15 mm, 25 mm, 30 mm or more, e.g., 50 mm; and a length of 5 mm ormore, e.g., 10 mm, 15 mm, 25 mm, 30 mm or more, e.g., 50 mm.

Referring now FIG. 7A-7D, pellets can be made so that they have a hollowinside. As shown, the hollow can be generally in-line with the center ofthe pellet (FIG. 7B), or out of line with the center of the pellet (FIG.7C). Making the pellet hollow inside can increase the rate ofdissolution in a liquid after irradiation.

Referring now to FIG. 7D, the pellet can have, e.g., a transverse shapethat is multi-lobal, e.g., tri-lobal as shown, or tetra-lobal,penta-lobal, hexa-lobal or deca-lobal. Making the pellets in suchtransverse shapes can also increase the rate of dissolution in asolution after irradiation.

Alternatively, the densified material can be in any other desired form,e.g., the densified material can be in the form of a mat, roll or bale.

Examples of Densification

In one example, half-gallon juice cartons made of un-printed white Kraftboard having a bulk density of 20 lb/ft³ can be used as a feedstock.Cartons can be folded flat and then fed into a shredder to produce aconfetti-like material having a width of between 0.1 inch and 0.5 inch,a length of between 0.25 inch and 1 inch and a thickness equivalent tothat of the starting material (about 0.075 inch). The confetti-likematerial can be fed to a rotary knife cutter, which shears theconfetti-like pieces, tearing the pieces apart and releasing fibrousmaterial.

In some cases, multiple shredder-shearer trains can be arranged inseries with output. In one embodiment, two shredder-shearer trains canbe arranged in series with output from the first shearer fed as input tothe second shredder. In another embodiment, three shredder-shearertrains can be arranged in series with output from the first shearer fedas input to the second shredder and output from the second shearer fedas input to the third shredder. Multiple passes through shredder-shearertrains are anticipated to increase decrease particle size and increaseoverall surface area within the feedstream.

In another example, fibrous material produced from shredding andshearing juice cartons can be treated to increase its bulk density. Insome cases, the fibrous material can be sprayed with water or a dilutestock solution of POLYOX™ WSR N10 (polyethylene oxide) prepared inwater. The wetted fibrous material can then be processed through apellet mill operating at room temperature. The pellet mill can increasethe bulk density of the feedstream by more than an order of magnitude.

Treatment

Pretreated biomass can be treated for use in primary productionprocesses by, for example, reducing the average molecular weight,crystallinity, and/or increasing the surface area and/or porosity of thebiomass. In some embodiments, the biomass can be treated to reduce therecalcitrance of the biomass. Treatment processes can include at leastone (e.g., one, two, three, four, or five) of irradiation, sonication,oxidation, pyrolysis, and steam explosion.

Recalcitrance is a term of art that, as used herein, broadly refers to abiomass material's resistance to the accessibility of polysaccharidedegrading agents (e.g., microorganisms and/or enzymes (e.g., microbialenzymes)) to polysaccharides contained within biomass (see, e.g., Himmelet al., National Renewable Energy Laboratory (NREL) Technical ReportNREL/TP-510-37902, August, 2005 and National Renewable Energy Laboratory(NREL) Technical Report NREL/BR-510-40742, March, 2007). For example,the accessibility of polysaccharides (e.g., cellulose and hemicellulose)in a first biomass material with a first recalcitrance level will belower than the accessibility of polysaccharides (e.g., cellulose andhemicellulose) in the same lignocellulosic material following treatmentto reduce the recalcitrance level of the material. In other words, thelevel of polysaccharides available to polysaccharide degrading agentswill be higher following treatment to reduce recalcitrance.

Assessing Recalcitrance Levels of Lignocellulosic Biomass

The recalcitrance level of a lignocellulosic material can be assessedusing a number of art-recognized methods. Examples of such methodsinclude, but are not limited to, surface characterization methods,enzymatic methods, and functional methods.

Exemplary surface characterization methods that can be used to assessthe recalcitrance level of lignocellulosic materials are known in theart (for a review see Himmel et al., National Renewable EnergyLaboratory (NREL) Technical Report NREL/TP-510-37902, August, 2005 andDing et al., Microscopy and Microanalysis, 14:1494-1495, 2004). Forexample, the recalcitrance level of lignocellulosic materials can beassessed using microscopic and/or spectroscopic surface analysis methods(e.g., using one or more of the surface analysis methods describedbelow) to identify, assess, and/or quantify changes (e.g., structuralchanges) in the lignocellulosic materials that indicate a reduction inthe recalcitrance of the material. Exemplary changes that can be used asindicia of a reduction in the recalcitrance of lignocellulosic materialsinclude the appearance of pitting or pores, and/or surface unwrapping ofmicrofibrils. See, for example, Himmel et al., National Renewable EnergyLaboratory (NREL) Technical Report NREL/TP-510-37902, August, 2005 andDing et al., Microscopy and Microanalysis, 14:1494-1495, 2004), whichdescribe the following methods:

(1) Scanning election microscopy (SEM) can be used to visualize thesurface morphology of biological and non-biological materials over awide range of magnifications (as high as 200,000× magnification) andwith high depth of field (see, e.g., Gomez et al., Biotechnology forBiofuels, 1, Oct. 23, 2008; Sivan et al., Appl. Microbiol. Biotechnol.,72:346-352, 2006). Typically, biological samples, such aslignocellulosic biomass samples, are coated with a thin layer ofelectron dense material, such as carbon or atomized gold, prior toanalysis. For example, samples can be mounted in SEM stubs and coatedwith gold/palladium. These mounted specimens can then be observed usingknown methods and devices, e.g., a JEOL JSM 6940LV SEM (Jeol Ltd.,Tokyo, Japan) at an accelerating voltage of 5 kV.

(2) More recently, methods have been developed for analyzing samplescontaining natural moisture, a technique referred to as environmentalmode SEM (ESEM), e.g., using the Quanta FEG 400 ESEM (FEI Company). Theuse of ESEM in the analysis of yeast cells is described by Ren et al.,Investigation of the morphology, viability and mechanical properties ofyeast cells in environmental SEM, Scanning, published online Aug. 5,2008). Such environmental mode methods can be used to analyzelignocellulosic biomass containing moisture without the use of highelectron dense coatings.

(3) Atomic force microscopy (AFM), e.g., using DI-Veeco MultiModePicoForce system (see, e.g., Stieg et al., Rev. Sci. Instrum.,79:103701, 2008) can also be used. AFM usefully allows analysis ofsurface topography at very high magnification while also allowinganalysis of the attractive and repulsive forces between the scanningprobe tip and the sample surface, thus providing height and phaseimages. AFM is being increasingly applied to the analysis of biologicalsamples due to its high atomic level resolution and its ease of use(samples do not require extensive sample preparation). In addition, AFMcan be used to observe dry and hydrated surfaces directly using atapping-probe.

(4) Transmission electron microscopy (TEM), e.g., using an FEI TecnaiF20, allows the determination of the internal structures of biologicaland non-biological materials up to at least 350,000× magnification.Typically, the determination of internal structures can be facilitatedusing shadowing techniques or staining with high contrast compounds.Compositional analysis of materials can also be performed by monitoringsecondary X-rays produced by the electron-specimen interaction usingenergy dispersive X-ray microanalysis. TEM-based methods for analyzingthe recalcitrance levels of a lignocellulosic material are described inthe art (see, e.g., Rhoads et al., Can. J. Microbiol., 41:592-600,1995).

(5) Near-field Scanning Optical Microscopy (NFSOM) using, e.g., aDI-Veeco Aurora-3 NSOM (Nikon), permits surfaces to be viewed with along depth of field light microscope that is adapted to conductsecondary spectrophotometric analysis such as UV/VIS, fluorescence, andlaser Raman. In some embodiments, NFSOM can be performed using anOlympus IX71 inverted microscope fitted with a DP70 high resolution CCDcamera to perform single molecule microscopy.

(6) Confocal microscopy (CFM) and confocal scanning laser microscopy(CSLM) (see, e.g., National Renewable Energy Laboratory (NREL) TechnicalReport NREL/BR-510-40742, March, 2007) can be used to generate opticalsections that can be used to build a three-dimensional image of asurface and internal structures. Typically, CFM and CSLM are performedin combination with labeling methods, for example fluorescent stains(see, e.g., Sole et al., Microb. Ecol., Published online on Nov. 4,2008).

In some embodiments, the recalcitrance level of a lignocellulosicmaterial can be assessed using one or more methods known in the art,e.g., methods described herein. The same sample, or a portion thereof,can then be assessed following treatment to observe a change (e.g., astructural change) in the recalcitrance. In some embodiments, theappearance or observance of pitting or pores, and/or surface unwrappingof microfibrils in or on a first lignocellulosic material with a firstrecalcitrance level will be less than the appearance or observance ofpitting or pores, and/or surface unwrapping of microfibrils in the samesample following treatment to reduce the recalcitrance level of thematerial.

Alternatively or in addition, a change (e.g., decrease) in therecalcitrance level of a lignocellulosic material can be analyzed usingenzymatic methods. For example, a lignocellulosic material can beincubated in the presence of one or more cellulases, e.g., before andafter treatment using the methods described herein. In some embodiments,an increase in the break down of cellulose by the cellulase indicates achange in the recalcitrant level of the material, e.g., a decrease inthe recalcitrance of the material. In some embodiments, the increase inthe break down of cellulose by the cellulase causes an increase in theamount of monosaccharide and/or disaccharides in the sample.

In some embodiments, the amount (e.g., concentration) of monosaccharidesand/or disaccharides resulting from the activity of an enzyme (e.g., acellulase) in a sample comprising a first lignocellulosic material witha first recalcitrance level will be lower than the amount (e.g.,concentration) of monosaccharide and/or disaccharides resulting from theactivity of an enzyme (e.g., a cellulase) in the same sample followingtreatment to reduce the recalcitrance level of the material.

Alternatively or in addition, a change (e.g., decrease) in therecalcitrance level of a lignocellulosic material can be analyzed usingfunctional methods. For example, a lignocellulosic material can becultured in the presence of a sugar fermenting microorganism, e.g.,using the culture methods disclosed herein, before and after treatmentusing the methods described herein. In some embodiments, an increase inthe level of the one or more products generated by the microorganismindicates a change in the recalcitrant level of the material, e.g., adecrease in the recalcitrance of the material.

In some embodiments, the growth rate of a microorganism and/or productgeneration by the microorganism in a sample comprising a firstlignocellulosic material with a first recalcitrance level will be lowerthan the growth rate of the microorganism and/or product generation bythe microorganism in the same sample following treatment to reduce therecalcitrance level of the material.

In some embodiments, a change in the recalcitrance level of a materialcan be expressed as; (1) a ratio (e.g., a measure of the recalcitrancelevel of a material prior to treatment versus a measure of therecalcitrance level or the material post-treatment); (2) a percentchange (e.g., decrease) in the recalcitrance level of a material; (3) apercent change (e.g., increase) in the level of polysaccharide availableto a polysaccharide degrading agent (e.g., an enzyme) after treatment,as compared to before the treatment, per weight measure of the startingbiomass material; or (4) a percent change (e.g., increase) in thesolubility of the material in a particular solvent.

In some instances, the second material has cellulose that has acrystallinity (^(T)C₂) that is lower than the crystallinity (^(T)C₁) ofthe cellulose of the first material. For example, (^(T)C₂) can be lowerthan (^(T)C₁) by more than about 10 percent, e.g., 15, 20, 25, 30, 35,40, or even more than about 50 percent.

In some embodiments, the starting crystallinity index (prior toirradiation) is from about 40 to about 87.5 percent, e.g., from about 50to about 75 percent or from about 60 to about 70 percent, and thecrystallinity index after irradiation is from about 10 to about 50percent, e.g., from about 15 to about 45 percent or from about 20 toabout 40 percent. However, in some embodiments, e.g., after extensiveirradiation, it is possible to have a crystallinity index of lower than5 percent. In some embodiments, the material after irradiation issubstantially amorphous.

In some embodiments, the starting number average molecular weight (priorto irradiation) is from about 200,000 to about 3,200,000, e.g., fromabout 250,000 to about 1,000,000 or from about 250,000 to about 700,000,and the number average molecular weight after irradiation is from about50,000 to about 200,000, e.g., from about 60,000 to about 150,000 orfrom about 70,000 to about 125,000. However, in some embodiments, e.g.,after extensive irradiation, it is possible to have a number averagemolecular weight of less than about 10,000 or even less than about5,000.

In some embodiments, the second material can have a level of oxidation(^(T)O₂) that is higher than the level of oxidation (^(T)O₁) of thefirst material. A higher level of oxidation of the material can aid inits dispersibility, swellability and/or solubility, further enhancingthe material's susceptibility to chemical, enzymatic, or biologicalattack. In some embodiments, to increase the level of the oxidation ofthe second material relative to the first material, the irradiation isperformed under an oxidizing environment, e.g., under a blanket of airor oxygen, producing a second material that is more oxidized than thefirst material. For example, the second material can have more hydroxylgroups, aldehyde groups, ketone groups, ester groups, or carboxylic acidgroups, which can increase its hydrophilicity.

Treatment Combinations

In some embodiments, biomass can be treated by applying at least one(e.g., two, three, four, or five) of the treatment methods describedherein, such as two or more of radiation, sonication, oxidation,pyrolysis, and steam explosion either with or without prior,intermediate, or subsequent biomass preparation as described herein. Thetreatment methods can be applied in any order, in multiples (e.g., twoor more applications of a treatment method), or concurrently to thebiomass, e.g., a cellulosic and/or lignocellulosic material. In otherembodiments, materials that include a carbohydrate are prepared byapplying three, four or more of any of the processes described herein(in any order or concurrently). For example, a carbohydrate can beprepared by applying radiation, sonication, oxidation, pyrolysis, and,optionally, steam explosion to a cellulosic and/or lignocellulosicmaterial (in any order or concurrently). The providedcarbohydrate-containing material can then be converted by one or moremicroorganisms, such as bacteria (e.g., gram positive bacteria, gramnegative bacteria, and extremophiles), yeast, or mixtures of yeast andbacteria, to a number of desirable products, as described herein.Multiple processes can provide materials that can be more readilyutilized by a variety of microorganisms because of their lower molecularweight, lower crystallinity, and/or enhanced solubility. Multipleprocesses can provide synergies and can reduce overall energy inputrequired in comparison to any single process.

For example, in some embodiments, biomass feedstocks can be providedthat include a carbohydrate that is produced by a process that includesirradiating and sonicating (in either order or concurrently) a biomassmaterial, a process that includes irradiating and oxidizing (in eitherorder or concurrently) a biomass material, a process that includesirradiating and pyrolyzing (in either order or concurrently) a biomassmaterial, a treatment process that includes irradiating and pyrolyzing(in either order or concurrently) a biomass material, or a process thatincludes irradiating and steam-exploding (in either order orconcurrently) a biomass material. The provided biomass feedstock canthen be contacted with a microorganism having the ability to convert atleast a portion, e.g., at least about 1 percent by weight, of thebiomass to the product.

In some embodiments, the process does not include hydrolyzing thebiomass, such as with an acid, base, and/or enzyme, e.g., a mineralacid, such as hydrochloric or sulfuric acid.

If desired, some or none of the biomass can include a hydrolyzedmaterial. For example, in some embodiments, at least about seventypercent by weight of the biomass is an unhydrolyzed material, e.g., atleast at 95 percent by weight of the feedstock is an unhydrolyzedmaterial. In some embodiments, substantially all of the biomass is anunhydrolyzed material. In some embodiments, 100% of the biomass isunhydrolyzed material.

Any feedstock or any reactor or fermentor charged with a feedstock caninclude a buffer, such as sodium bicarbonate, ammonium chloride or Tris;an electrolyte, such as potassium chloride, sodium chloride, or calciumchloride; a growth factor, such as biotin and/or a base pair such asuracil or an equivalent thereof; a surfactant, such as Tween® orpolyethylene glycol; a mineral, such as such as calcium, chromium,copper, iodine, iron, selenium, or zinc; or a chelating agent, such asethylene diamine, ethylene diamine tetraacetic acid (EDTA) (or its saltform, e.g., sodium or potassium EDTA), or dimercaprol.

When radiation is utilized as or in the treatment, it can be applied toany sample that is dry or wet, or even dispersed in a liquid, such aswater. For example, irradiation can be performed on biomass material inwhich less than about 25 percent by weight of the biomass material hassurfaces wetted with a liquid, such as water. In some embodiments,irradiating is performed on biomass material in which substantially noneof the biomass material is wetted with a liquid, such as water.

In some embodiments, any processing described herein occurs after thebiomass material remains dry as acquired or has been dried, e.g., usingheat and/or reduced pressure. For example, in some embodiments, thebiomass material has less than about five percent by weight retainedwater, measured at 25° C. and at fifty percent relative humidity.

If desired, a swelling agent, as defined herein, can be utilized in anyprocess described herein. In some embodiments, when a biomass materialis processed using radiation, less than about 25 percent by weight ofthe biomass material is in a swollen state, the swollen state beingcharacterized as having a volume of more than about 2.5 percent higherthan an unswollen state, e.g., more than 5.0, 7.5, 10, or 15 percenthigher than the unswollen state. In some embodiments, when radiation isutilized on a biomass material, substantially none of the biomassmaterial is in a swollen state.

In specific embodiments when radiation is utilized, the biomass materialincludes a swelling agent, and swollen biomass material receives a doseof less than about 10 Mrad.

When radiation is utilized in any process, it can be applied while thebiomass is exposed to air, oxygen-enriched air, or even oxygen itself,or blanketed by an inert gas such as nitrogen, argon, or helium. Whenmaximum oxidation is desired, an oxidizing environment is utilized, suchas air or oxygen.

When radiation is utilized, it can be applied to biomass under apressure of greater than about 2.5 atmospheres, such as greater than 5,10, 15, 20 or even greater than about 50 atmospheres. Irradiation canincrease the solubility, swellability, or dispersibility of the biomassin a solvent.

In specific embodiments, the process includes irradiating and sonicatingand irradiating precedes sonicating. In other specific embodiments,sonication precedes irradiating, or irradiating and sonicating occursubstantially concurrently.

In some embodiments, the process includes irradiating and sonicating (ineither order or concurrently) and further includes oxidizing, pyrolyzingor steam exploding.

When the process includes radiation, the irradiating can be performedutilizing an ionizing radiation, such as gamma rays, x-rays, energeticultraviolet radiation, such as ultraviolet C radiation having awavelength of from about 100 nm to about 280 nm, a beam of particles,such as a beam of electrons, slow neutrons or alpha particles. In someembodiments, irradiating includes two or more radiation sources, such asgamma rays and a beam of electrons, which can be applied in either orderor concurrently.

In specific embodiments, sonicating can performed at a frequency ofbetween about 15 kHz and about 25 kHz, such as between about 18 kHz and22 kHz utilizing a 1 KW or larger horn, e.g., a 2, 3, 4, 5, or even a 10KW horn.

In some embodiments, the biomass has a first number average molecularweight and the resulting carbohydrate includes a second cellulose havinga second number average molecular weight lower than the first numberaverage molecular weight. For example, the second number averagemolecular weight is lower than the first number average molecular weightby more than about twenty-five percent, e.g., 2×, 3×, 5×, 7×, 10×, 25×,even 100× reduction.

In some embodiments, the first cellulose has a first crystallinity andthe second cellulose has a second crystallinity lower than the firstcrystallinity, such as lower than about two, three, five, ten, fifteenor twenty-five percent lower.

In some embodiments, the first cellulose has a first level of oxidationand the second cellulose has a second level of oxidation higher than thefirst level of oxidation, such as two, three, four, five, ten or eventwenty-five percent higher.

In some embodiments, the first biomass has a first level ofrecalcitrance and the resulting biomass has a second level ofrecalcitrance that is lower than the first level.

Radiation Treatment

One or more irradiation processing sequences can be used to processbiomass from a wide variety of different sources to extract usefulsubstances from the feedstock, and to provide partially degraded organicmaterial which functions as input to further processing steps and/orsequences. Irradiation can reduce the recalcitrance, molecular weightand/or crystallinity of feedstock.

In some embodiments, energy deposited in a material that releases anelectron from its atomic orbital is used to irradiate the materials. Theradiation can be provided by 1) heavy charged particles, such as alphaparticles or protons, 2) electrons, produced, for example, in beta decayor electron beam accelerators, or 3) electromagnetic radiation, forexample, gamma rays, x rays, or ultraviolet rays. In one approach,radiation produced by radioactive substances can be used to irradiatethe feedstock. In some embodiments, any combination in any order orconcurrently of (1) through (3) can be utilized. In another approach,electromagnetic radiation (e.g., produced using electron beam emitters)can be used to irradiate the feedstock. The doses applied depend on thedesired effect and the particular feedstock. For example, high doses ofradiation can break chemical bonds within feedstock components and lowdoses of radiation can increase chemical bonding (e.g., cross-linking)within feedstock components. In some instances when chain scission isdesirable and/or polymer chain functionalization is desirable, particlesheavier than electrons, such as protons, helium nuclei, argon ions,silicon ions, neon ions, carbon ions, phosphorus ions, oxygen ions ornitrogen ions can be utilized. When ring-opening chain scission isdesired, positively charged particles can be utilized for their Lewisacid properties for enhanced ring-opening chain scission.

Referring to FIG. 8, in one method, a first material 2 that is orincludes cellulose having a first number average molecular weight(^(T)M_(N1)) is irradiated, e.g., by treatment with ionizing radiation(e.g., in the form of gamma radiation, X-ray radiation, 100 nm to 280 nmultraviolet (UV) light, a beam of electrons or other charged particles)to provide a second material 3 that includes cellulose having a secondnumber average molecular weight (^(T)M_(N2)) lower than the first numberaverage molecular weight. The second material (or the first and secondmaterial) can be combined with a microorganism (e.g., a bacterium or ayeast) that can utilize the second and/or first material to produce aproduct 5.

Since the second material 3 has cellulose having a reducedrecalcitrance, molecular weight relative to the first material, and insome instances, a reduced crystallinity, the second material isgenerally more dispersible, swellable and/or soluble in a solutioncontaining a microorganism. These properties make the second material 3more susceptible to chemical, enzymatic and/or biological attack (e.g.,by a microorganism) relative to the first material 2, which can greatlyimprove the production rate and/or production level of a desiredproduct, e.g., ethanol. Radiation can also sterilize the materials.

In some embodiments, the second number average molecular weight (M_(N2))is lower than the first number average molecular weight (^(T)M_(N1)) bymore than about 10 percent, e.g., 15, 20, 25, 30, 35, 40, 50 percent, 60percent, or even more than about 75 percent.

Ionizing Radiation

Each form of radiation ionizes the biomass via particular interactions,as determined by the energy of the radiation. Heavy charged particlesprimarily ionize matter via Coulomb scattering; furthermore, theseinteractions produce energetic electrons that can further ionize matter.Alpha particles are identical to the nucleus of a helium atom and areproduced by the alpha decay of various radioactive nuclei, such asisotopes of bismuth, polonium, astatine, radon, francium, radium,several actinides, such as actinium, thorium, uranium, neptunium,curium, californium, americium, and plutonium.

When particles are utilized, they can be neutral (uncharged), positivelycharged or negatively charged. When charged, the charged particles canbear a single positive or negative charge, or multiple charges, e.g.,one, two, three or even four or more charges. In instances in whichchain scission is desired, positively charged particles can bedesirable, in part, due to their acidic nature. When particles areutilized, the particles can have the mass of a resting electron, orgreater, e.g., 500, 1000, 1500, or 2000 or more times the mass of aresting electron. For example, the particles can have a mass of fromabout 1 atomic units to about 150 atomic units, e.g., from about 1atomic units to about 50 atomic units, or from about 1 to about 25,e.g., 1, 2, 3, 4, 5, 10, 12 or 15 amu. Accelerators used to acceleratethe particles can be electrostatic DC, electrodynamic DC, RF linear,magnetic induction linear or continuous wave. For example, cyclotrontype accelerators are available from IBA, Belgium, such as theRhodotron® system, while DC type accelerators are available from RDI,now IBA Industrial, such as the Dynamitron®. Ions and ion acceleratorsare discussed in Introductory Nuclear Physics, Kenneth S. Krane, JohnWiley & Sons, Inc. (1988), Krsto Prelec, FIZIKA B 6 (1997) 4, 177-206,Chu, William T., “Overview of Light-Ion Beam Therapy”, Columbus-Ohio,ICRU-IAEA Meeting, 18-20 Mar. 2006, Iwata, Y. et al.,“Alternating-Phase-Focused IH-DTL for Heavy-Ion Medical Accelerators”,Proceedings of EPAC 2006, Edinburgh, Scotland, and Leitner, C. M. etal., “Status of the Superconducting ECR Ion Source Venus”, Proceedingsof EPAC 2000, Vienna, Austria. Typically, generators are housed in avault, e.g., of lead or concrete.

Electrons interact via Coulomb scattering and bremsstrahlung radiationproduced by changes in the velocity of electrons. Electrons can beproduced by radioactive nuclei that undergo beta decay, such as isotopesof iodine, cesium, technetium, and iridium. Alternatively, an electrongun can be used as an electron source via thermionic emission.

Electromagnetic radiation interacts via three processes: photoelectricabsorption, Compton scattering, and pair production. The dominatinginteraction is determined by the energy of the incident radiation andthe atomic number of the material. The summation of interactionscontributing to the absorbed radiation in cellulosic material can beexpressed by the mass absorption coefficient (see “Ionization Radiation”in PCT/US2007/022719).

Electromagnetic radiation is subclassified as gamma rays, x rays,ultraviolet rays, infrared rays, microwaves, or radio waves, dependingon its wavelength.

For example, gamma radiation can be employed to irradiate the materials.Referring to FIGS. 9 and 10 (an enlarged view of region R), a gammairradiator 10 includes gamma radiation sources 408, e.g., ⁶⁰Co pellets,a working table 14 for holding the materials to be irradiated andstorage 16, e.g., made of a plurality of iron plates, all of which arehoused in a concrete containment chamber (vault) 20 that includes a mazeentranceway 22 beyond a lead-lined door 26. Storage 16 includes aplurality of channels 30, e.g., sixteen or more channels, allowing thegamma radiation sources to pass through storage on their way proximatethe working table.

In operation, the sample to be irradiated is placed on a working table.The irradiator is configured to deliver the desired dose rate andmonitoring equipment is connected to an experimental block 31. Theoperator then leaves the containment chamber, passing through the mazeentranceway and through the lead-lined door. The operator mans a controlpanel 32, instructing a computer 33 to lift the radiation sources 12into working position using cylinder 36 attached to a hydraulic pump 40.

Gamma radiation has the advantage of a significant penetration depthinto a variety of material in the sample. Sources of gamma rays includeradioactive nuclei, such as isotopes of cobalt, calcium, technicium,chromium, gallium, indium, iodine, iron, krypton, samarium, selenium,sodium, thalium, and xenon.

Sources of x rays include electron beam collision with metal targets,such as tungsten or molybdenum or alloys, or compact light sources, suchas those produced commercially by Lyncean. Sources for ultravioletradiation include deuterium or cadmium lamps. Sources for infraredradiation include sapphire, zinc, or selenide window ceramic lamps.Sources for microwaves include klystrons, Slevin type RF sources, oratom beam sources that employ hydrogen, oxygen, or nitrogen gases.

Various other irradiating devices may be used in the methods disclosedherein, including field ionization sources, electrostatic ionseparators, field ionization generators, thermionic emission sources,microwave discharge ion sources, recirculating or static accelerators,dynamic linear accelerators, van de Graaff accelerators, and foldedtandem accelerators. Such devices are disclosed, for example, in U.S.Provisional Application Ser. No. 61/073,665 (which served as priorityfor U.S. Pat. No. 8,025,098 to Medoff), the complete disclosure of whichis incorporated herein by reference.

Electron Beam

In some embodiments, a beam of electrons is used as the radiationsource. A beam of electrons has the advantages of high dose rates (e.g.,1, 5, or even 10 Mrad per second), high throughput, less containment,and less confinement equipment. Electrons can also be more efficient atcausing chain scission. In addition, electrons having energies of 4-10MeV can have a penetration depth of 5 to 30 mm or more, such as 40 mm.

Electron beams can be generated, e.g., by electrostatic generators,cascade generators, transformer generators, low energy accelerators witha scanning system, low energy accelerators with a linear cathode, linearaccelerators, and pulsed accelerators. Electrons as an ionizingradiation source can be useful, e.g., for relatively thin piles ofmaterials, e.g., less than 0.5 inch, e.g., less than 0.4 inch, 0.3 inch,0.2 inch, or less than 0.1 inch. In some embodiments, the energy of eachelectron of the electron beam is from about 0.3 MeV to about 2.0 MeV(million electron volts), e.g., from about 0.5 MeV to about 1.5 MeV, orfrom about 0.7 MeV to about 1.25 MeV.

FIG. 11 shows a process flow diagram 3000 that includes various steps inan electron beam irradiation feedstock pretreatment sequence. In firststep 3010, a supply of dry feedstock is received from a feed source. Asdiscussed above, the dry feedstock from the feed source can bepre-processed prior to delivery to the electron beam irradiationdevices. For example, if the feedstock is derived from plant sources,certain portions of the plant material can be removed prior tocollection of the plant material and/or before the plant material isdelivered by the feedstock transport device. Alternatively, or inaddition, as expressed in optional step 3020, the biomass feedstock canbe subjected to mechanical processing (e.g., to reduce the averagelength of fibers in the feedstock) prior to delivery to the electronbeam irradiation devices.

In step 3030, the dry feedstock is transferred to a feedstock transportdevice (e.g., a conveyor belt) and is distributed over thecross-sectional width of the feedstock transport device approximatelyuniformly by volume. This can be accomplished, for example, manually orby inducing a localized vibration motion at some point in the feedstocktransport device prior to the electron beam irradiation processing.

In some embodiments, a mixing system introduces a chemical agent 3045into the feedstock in an optional process 3040 that produces a slurry.Combining water with the processed feedstock in mixing step 3040 createsan aqueous feedstock slurry that can be transported through, forexample, piping rather than using, for example, a conveyor belt.

The next step 3050 is a loop that encompasses exposing the feedstock (indry or slurry form) to electron beam radiation via one or more (say, N)electron beam irradiation devices. The feedstock slurry is moved througheach of the N “showers” of electron beams at step 3052. The movement caneither be at a continuous speed through and between the showers, orthere can be a pause through each shower, followed by a sudden movementto the next shower. A small slice of the feedstock slurry is exposed toeach shower for some predetermined exposure time at step 3053.

Electron beam irradiation devices can be procured commercially from IonBeam Applications, Louvain-la-Neuve, Belgium or the Titan Corporation,San Diego, Calif. Typical electron energies can be 1 MeV, 2 MeV, 4.5MeV, 7.5 MeV, or 10 MeV. Typical electron beam irradiation device powercan be 1 kW, 5 kW, 10 kW, 20 kW, 50 kW, 100 kW, 250 kW, or 500 kW.Effectiveness of depolymerization of the feedstock slurry depends on theelectron energy used and the dose applied, while exposure time dependson the power and dose. Typical doses can take values of 1 kGy, 5 kGy, 10kGy, 20 kGy, 50 kGy, 100 kGy, or 200 kGy.

Tradeoffs in considering electron beam irradiation device powerspecifications include cost to operate, capital costs, depreciation, anddevice footprint. Tradeoffs in considering exposure dose levels ofelectron beam irradiation would be energy costs and environment, safety,and health (ESH) concerns. Tradeoffs in considering electron energiesinclude energy costs; here, a lower electron energy can be advantageousin encouraging depolymerization of certain feedstock slurry (see, forexample, Bouchard, et al, Cellulose (2006) 13: 601-610).

It can be advantageous to provide a double-pass of electron beamirradiation in order to provide a more effective depolymerizationprocess. For example, the feedstock transport device could direct thefeedstock (in dry or slurry form) underneath and in a reverse directionto its initial transport direction. Double-pass systems can allowthicker feedstock slurries to be processed and can provide a moreuniform depolymerization through the thickness of the feedstock slurry.

The electron beam irradiation device can produce either a fixed beam ora scanning beam. A scanning beam can be advantageous with large scansweep length and high scan speeds, as this would effectively replace alarge, fixed beam width. Further, available sweep widths of 0.5 m, 1 m,2 m or more are available. One suitable device is referenced in Example22.

Once a portion of feedstock slurry has been transported through the Nelectron beam irradiation devices, it can be necessary in someembodiments, as in step 3060, to mechanically separate the liquid andsolid components of the feedstock slurry. In these embodiments, a liquidportion of the feedstock slurry is filtered for residual solid particlesand recycled back to the slurry preparation step 3040. A solid portionof the feedstock slurry is then advanced on to the next processing step3070 via the feedstock transport device. In other embodiments, thefeedstock is maintained in slurry form for further processing.

Heavy Ion Particle Beams

Particles heavier than electrons can be utilized to irradiatecarbohydrates or materials that include carbohydrates, e.g., cellulosicmaterials, lignocellulosic materials, starchy materials, or mixtures ofany of these and others described herein. For example, protons, heliumnuclei, argon ions, silicon ions, neon ions, carbon ions, phosphorusions, oxygen ions or nitrogen ions can be utilized. In some embodiments,particles heavier than electrons can induce higher amounts of chainscission. In some instances, positively charged particles can inducehigher amounts of chain scission than negatively charged particles dueto their acidity.

Heavier particle beams can be generated, e.g., using linear acceleratorsor cyclotrons. In some embodiments, the energy of each particle of thebeam is from about 1.0 MeV/atomic unit to about 6,000 MeV/atomic unit,e.g., from about 3 MeV/atomic unit to about 4,800 MeV/atomic unit, orfrom about 10 MeV/atomic unit to about 1,000 MeV/atomic unit.

Electromagnetic Radiation

In embodiments in which the irradiating is performed withelectromagnetic radiation, the electromagnetic radiation can have, e.g.,energy per photon (in electron volts) of greater than 10² eV, e.g.,greater than 10³, 10⁴, 10⁵, 10⁶, or even greater than 10⁷ eV. In someembodiments, the electromagnetic radiation has energy per photon ofbetween 10⁴ and 10⁷, e.g., between 10⁵ and 10⁶ eV. The electromagneticradiation can have a frequency of, e.g., greater than 10¹⁶ Hz, greaterthan 10¹⁷ Hz, 10 ¹⁸, 10¹⁹, 10²⁰, or even greater than 10²¹ Hz. In someembodiments, the electromagnetic radiation has a frequency of between10¹⁸ and 10²² Hz, e.g., between 10¹⁹ to 10²¹ Hz.

Doses

In some embodiments, the irradiating (with any radiation source or acombination of sources) is performed until the material receives a doseof at least 0.25 Mrad, e.g., at least 1.0 Mrad, at least 2.5 Mrad, atleast 5.0 Mrad, or at least 10.0 Mrad. In some embodiments, theirradiating is performed until the material receives a dose of between1.0 Mrad and 6.0 Mrad, e.g., between 1.5 Mrad and 4.0 Mrad.

In some embodiments, the irradiating is performed at a dose rate ofbetween 5.0 and 1500.0 kilorads/hour, e.g., between 10.0 and 750.0kilorads/hour or between 50.0 and 350.0 kilorads/hours.

In some embodiments, two or more radiation sources are used, such as twoor more ionizing radiations. For example, samples can be treated, in anyorder, with a beam of electrons, followed by gamma radiation and UVlight having wavelengths from about 100 nm to about 280 nm. In someembodiments, samples are treated with three ionizing radiation sources,such as a beam of electrons, gamma radiation, and energetic UV light.

Alternatively, in another example, a fibrous biomass material thatincludes a cellulosic and/or lignocellulosic material is irradiated and,optionally, treated with acoustic energy, e.g., ultrasound.

In one example of the use of radiation as a treatment, half-gallon juicecartons made of un-printed polycoated white Kraft board having a bulkdensity of 20 lb/ft³ are used as a feedstock. Cartons are folded flatand then fed into a sequence of three shredder-shearer trains arrangedin series with output from the first shearer fed as input to the secondshredder, and output from the second shearer fed as input to the thirdshredder. The fibrous material produced by the can be sprayed with waterand processed through a pellet mill operating at room temperature. Thedensified pellets can be placed in a glass ampoule which is evacuatedunder high vacuum and then back-filled with argon gas. The ampoule issealed under argon. The pellets in the ampoule are irradiated with gammaradiation for about 3 hours at a dose rate of about 1 Mrad per hour toprovide an irradiated material in which the cellulose has a lowermolecular weight than the starting material.

Quenching and Controlled Functionalization of Biomass

After treatment with one or more ionizing radiations, such as photonicradiation (e.g., X-rays or gamma-rays), e-beam radiation or particlesheavier than electrons that are positively or negatively charged (e.g.,protons or carbon ions), any of the carbohydrate-containing materials ormixtures described herein become ionized; that is, they include radicalsat levels that are detectable with an electron spin resonancespectrometer. The current limit of detection of the radicals is about10¹⁴ spins at room temperature. After ionization, any biomass materialthat has been ionized can be quenched to reduce the level of radicals inthe ionized biomass, e.g., such that the radicals are no longerdetectable with the electron spin resonance spectrometer. For example,the radicals can be quenched by the application of a sufficient pressureto the biomass and/or utilizing a fluid in contact with the ionizedbiomass, such as a gas or liquid, that reacts with (quenches) theradicals. Using a gas or liquid to at least aid in the quenching of theradicals can be used to functionalize the ionized biomass with a desiredamount and kinds of functional groups, such as carboxylic acid groups,enol groups, aldehyde groups, nitro groups, nitrile groups, aminogroups, alkyl amino groups, alkyl groups, chloroalkyl groups orchlorofluoroalkyl groups. In some instances, such quenching can improvethe stability of some of the ionized biomass materials. For example,quenching can improve the biomass's resistance to oxidation.Functionalization by quenching can also improve the solubility of anybiomass described herein, can improve its thermal stability, which canimprove material utilization by various microorganisms. For example, thefunctional groups imparted to the biomass material by the quenching canact as receptor sites for attachment by microorganisms, e.g., to enhancecellulose hydrolysis by various microorganisms.

FIG. 11A illustrates changing a molecular and/or a supramolecularstructure of a biomass feedstock by pretreating the biomass feedstockwith ionizing radiation, such as with electrons or ions of sufficientenergy to ionize the biomass feedstock, to provide a first level ofradicals. As shown in FIG. 11A, if it ionized biomass remains in theatmosphere, it will be oxidized, such as to an extent that carboxylicacid groups are generated by reacting with the atmospheric oxygen. Insome instances with some materials, such oxidation is desired because itcan aid in the further breakdown in molecular weight of thecarbohydrate-containing biomass, and the oxidation groups, e.g.,carboxylic acid groups can be helpful for solubility and microorganismutilization in some instances. However, since the radicals can “live”for some time after irradiation, e.g., longer than 1 day, 5 days, 30days, 3 months, 6 months or even longer than 1 year, materialsproperties can continue to change over time, which in some instances,can be undesirable. Detecting radicals in irradiated samples by electronspin resonance spectroscopy and radical lifetimes in such samples isdiscussed in Bartolotta et al., Physics in Medicine and Biology, 46(2001), 461-471 and in Bartolotta et al., Radiation ProtectionDosimetry, Vol. 84, Nos. 1-4, pp. 293-296 (1999). As shown in FIG. 11A,the ionized biomass can be quenched to functionalize and/or to stabilizethe ionized biomass. At any point, e.g., when the material is “alive”(still has a substantial quantity of reactive intermediates such asradicals), “partially alive” or fully quenched, the treated biomass canbe converted into a product, e.g., a food.

In some embodiments, the quenching includes an application of pressureto the biomass, such as by mechanically deforming the biomass, e.g.,directly mechanically compressing the biomass in one, two, or threedimensions, or applying pressure to a fluid in which the biomass isimmersed, e.g., isostatic pressing. In such instances, the deformationof the material itself brings radicals, which are often trapped incrystalline domains, in close enough proximity so that the radicals canrecombine, or react with another group. In some instances, the pressureis applied together with the application of heat, such as a sufficientquantity of heat to elevate the temperature of the biomass to above amelting point or softening point of a component of the biomass, such aslignin, cellulose or hemicellulose. Heat can improve molecular mobilityin the polymeric material, which can aid in the quenching of theradicals. When pressure is utilized to quench, the pressure can begreater than about 1000 psi, such as greater than about 1250 psi, 1450psi, 3625 psi, 5075 psi, 7250 psi, 10000 psi or even greater than 15000psi.

In some embodiments, quenching includes contacting the biomass with afluid, such as a liquid or gas, e.g., a gas capable of reacting with theradicals, such as acetylene or a mixture of acetylene in nitrogen,ethylene, chlorinated ethylenes or chlorofluoroethylenes, propylene ormixtures of these gases. In other particular embodiments, quenchingincludes contacting the biomass with a liquid, e.g., a liquid solublein, or at least capable of penetrating into the biomass and reactingwith the radicals, such as a diene, such as 1,5-cyclooctadiene. In somespecific embodiments, the quenching includes contacting the biomass withan antioxidant, such as Vitamin E. If desired, the biomass feedstock caninclude an antioxidant dispersed therein, and the quenching can comefrom contacting the antioxidant dispersed in the biomass feedstock withthe radicals. Combinations of these and other quenching materials can beused.

Other methods for quenching are possible. For example, any method forquenching radicals in polymeric materials described in Muratoglu et al.,U.S. Patent Application Publication No. 2008/0067724 and Muratoglu etal., U.S. Pat. No. 7,166,650, can be utilized for quenching any ionizedbiomass material described herein. Furthermore any quenching agent(described as a “sensitizing agent” in the above-noted Muratogludisclosures) and/or any antioxidant described in either Muratoglureference can be utilized to quench any ionized biomass material.

Functionalization can be enhanced by utilizing heavy charged ions, suchas any of the heavier ions described herein. For example, if it isdesired to enhance oxidation, charged oxygen ions can be utilized forthe irradiation. If nitrogen functional groups are desired, nitrogenions or ions that includes nitrogen can be utilized. Likewise, if sulfuror phosphorus groups are desired, sulfur or phosphorus ions can be usedin the irradiation.

In some embodiments, after quenching any of the quenched ionizedmaterials described herein can be further treated with one or more ofradiation, such as ionizing or non-ionizing radiation, sonication,pyrolysis, and oxidation for additional molecular and/or supramolecularstructure change.

Particle Beam Exposure in Fluids

In some cases, the cellulosic or lignocellulosic materials can beexposed to a particle beam in the presence of one or more additionalfluids (e.g., gases and/or liquids). Exposure of a material to aparticle beam in the presence of one or more additional fluids canincrease the efficiency of the treatment.

In some embodiments, the material is exposed to a particle beam in thepresence of a fluid such as air. Particles accelerated in any one ormore of the types of accelerators disclosed herein (or another type ofaccelerator) are coupled out of the accelerator via an output port(e.g., a thin membrane such as a metal foil), pass through a volume ofspace occupied by the fluid, and are then incident on the material. Inaddition to directly treating the material, some of the particlesgenerate additional chemical species by interacting with fluid particles(e.g., ions and/or radicals generated from various constituents of air,such as ozone and oxides of nitrogen). These generated chemical speciescan also interact with the material, and can act as initiators for avariety of different chemical bond-breaking reactions in the material.For example, any oxidant produced can oxidize the material, which canresult in molecular weight reduction.

In certain embodiments, additional fluids can be selectively introducedinto the path of a particle beam before the beam is incident on thematerial. As discussed above, reactions between the particles of thebeam and the particles of the introduced fluids can generate additionalchemical species, which react with the material and can assist infunctionalizing the material, and/or otherwise selectively alteringcertain properties of the material. The one or more additional fluidscan be directed into the path of the beam from a supply tube, forexample. The direction and flow rate of the fluid(s) that is/areintroduced can be selected according to a desired exposure rate and/ordirection to control the efficiency of the overall treatment, includingeffects that result from both particle-based treatment and effects thatare due to the interaction of dynamically generated species from theintroduced fluid with the material. In addition to air, exemplary fluidsthat can be introduced into the ion beam include oxygen, nitrogen, oneor more noble gases, one or more halogens, and hydrogen.

Irradiating Low Bulk Density Biomass Materials and Cooling IrradiatedBiomass

During treatment of biomass materials with ionizing radiation,especially at high dose rates, such as at rates greater then 0.15 Mradper second, e.g., 0.25 Mrad/s, 0.35 Mrad/s, 0.5 Mrad/s, 0.75 Mrad/s oreven greater than 1 Mrad/sec, biomass materials can retain significantquantities of heat so that the temperature of the biomass materialsbecome elevated. While higher temperatures can, in some embodiments, beadvantageous, e.g., when a faster reaction rate is desired, it isadvantageous to control the heating of the biomass to retain controlover the chemical reactions initiated by the ionizing radiation, such ascross-linking, chain scission and/or grafting, e.g., to maintain processcontrol. Low bulk density materials, such as those having a bulk densityof less than about 0.4 g/cm³, e.g., less than about 0.35, 0.25 or lessabout 0.15 g/cm³, especially when combined with materials that have thincross-sections, such as fibers having small transverse dimensions, aregenerally easier to cool. In addition, photons and particles cangenerally penetrate further into and through materials having arelatively low bulk density, which can allow for the processing oflarger volumes of materials at higher rates, and can allow for the useof photons and particles that having lower energies, e.g., 0.25 Mev, 0.5MeV, 0.75 MeV or 1.0 MeV, which can reduce safety shieldingrequirements. Many of the biomass materials described herein can beprocessed in one or more of the systems shown in FIGS. 11B, 11C, 11D and11E, which are described below. The systems shown allow one or moretypes of ionizing radiation, such as relativistic electrons or electronsin combination with X-rays, to be applied to low bulk density biomassmaterials at highs dose rates, such as at a rate greater than 1.0, 1.5,2.5 Mrad/s or even greater than about 5.0 Mrad/s, and then to allow forcooling of the biomass prior to applying radiation for a second, third,fourth, fifth, sixth, seventh, eighth, ninth or even a tenth time.

For example, in one method of changing a molecular and/or asupramolecular structure of a biomass feedstock, the biomass ispretreated at a first temperature with ionizing radiation, such asphotons, electrons or ions (e.g., singularly or multiply charged cationsor anions), for a sufficient time and/or a sufficient dose to elevatethe biomass feedstock to a second temperature higher than the firsttemperature. The pretreated biomass is then cooled below the secondtemperature. Finally, if desired, the cooled biomass can be treated oneor more times with radiation, e.g., with ionizing radiation. If desired,cooling can be applied to the biomass after and/or during each radiationtreatment.

In some embodiments, the cooling of the biomass feedstock is to anextent that, after cooling, the biomass is at a third temperature belowthe first temperature.

For example, and as will be explained in more detail below, treatingbiomass feedstock with the ionizing radiation can be performed as thebiomass feedstock is being pneumatically conveyed in a fluid, such as ain a gas, such as nitrogen or air. To aid in molecular weight breakdownand/or functionalization of the materials, the gas can be saturated withany swelling agent described herein and/or water vapor. For example,acidic water vapor can be utilized. To aid in molecular weightbreakdown, the water can be acidified with an organic acid, such asformic, or acetic acid, or a mineral acid, such as sulfuric orhydrochloric acid.

For example, and as will be explained in more detail below, the treatingbiomass feedstock with the ionizing radiation can be performed as thebiomass feedstock falls under the influence of gravity. This procedurecan effectively reduce the bulk density of the biomass feedstock as itis being processed and can aid in the cooling of the biomass feedstock.For example, the biomass can be conveyed from a first belt at a firstheight above the ground and then can be captured by a second belt at asecond level above the ground lower than the first level. For example,in some embodiments, the trailing edge of the first belt and the leadingedge of the second belt defining a gap. Advantageously, the ionizingradiation, such as a beam of electrons, protons, or other ions, can beapplied at the gap to prevent damage to the biomass conveyance system.

In the methods described herein, cooling of the biomass can includecontacting the biomass with a fluid, such as a gas, at a temperaturebelow the first or second temperature, such as gaseous nitrogen at orabout 77 K. Even water, such as water at a temperature below nominalroom temperature (e.g., 25 degrees Celsius) can be utilized.

The biomass feedstock can be treated at a first temperature withionizing radiation for a sufficient time and/or a sufficient dose, suchas from about 1 second to about 10 seconds at a dose rate of about 0.5Mrad/s to about 5 Mrad/s, to elevate the biomass feedstock to a secondtemperature higher than the first temperature. After applying theradiation, the biomass can be cooled below the second temperature. Thecooled treated biomass is treated with radiation, such as an ionizingradiation, and then the treated biomass is contacted with amicroorganism having the ability to convert at least a portion, e.g., atleast about 1 percent by weight, of the biomass to the product.

In some embodiments, a method of changing a molecular and/or asupramolecular structure of a biomass feedstock includes optionally,pretreating the biomass feedstock by reducing one or more dimensions ofindividual pieces of the biomass feedstock and applying ionizingradiation, such as photons, electrons or ions, to the biomass feedstock.In such embodiments, the biomass feedstock to which the ionizingradiation is applied has a bulk density of less than about 0.35 g/cm³,such as less than about 0.3, 0.25, 0.20, or less than about 0.15 g/cm³during the application of the ionizing radiation. In such embodiments,the biomass feedstock can be cooled, and then ionizing radiation can beapplied to the cooled biomass. In some advantageous embodiments, thebiomass feedstock is or includes discrete fibers and/or particles havinga maximum dimension of not more than about 0.5 mm, such as not more thanabout 0.25 mm, not more than about 0.1 mm, not more than about 0.05 mm,or not more than about 0.025 mm.

Referring particularly now to FIGS. 11B and 11C, which shows a biomassmaterial generating, treating, conveying, and irradiating device 1170(shielding not illustrated in the drawings). In operation, paper sheet1173, e.g., scrap bleached Kraft paper sheet, is supplied from a roll1172 and delivered to a fiberizing apparatus 1174, such as a rotaryshearer. The sheet 1173 is converted into fibrous material 1112 and isdelivered to a fiber-loading zone 1180 by conveyer 1178. If desired, thefibers of the fibrous material can be separated, e.g., by screening,into fractions having different L/D ratios. In some embodiments, thefibrous material 1112 of generally a low bulk density and advantageouslythin cross-sections, is delivered continuously to zone 1180, and inother embodiments, the fibrous material is delivered in batches. Ablower 1182 in loop 1184 is positioned adjacent to the fiber-loadingzone 1180 and is capable of moving a fluid medium, e.g., air, at avelocity and volume sufficient to pneumatically circulate the fibrousmaterial 1112 in a direction indicated by arrow 1188 through loop 1184.

In some embodiments, the velocity of air traveling in the loop issufficient to uniformly disperse and transport the fibrous materialaround the entire loop 1184. In some embodiments, the velocity of flowis greater than 2,500 feet/minute, e.g., 5,000 feet/minute, 6,000feet/minute or more, e.g., 7,500 feet/minute or 8,500 feet/minute.

The entrained fibrous material 1112 traversing the loop passes anapplication zone 1190, which forms part of loop 1184. Here, any desiredadditives described herein are applied, such as a liquid, such as water,such as acidified or water made basic. In operation, application zone1190 applies an additive, such as a liquid solution 1196 to thecirculating fibrous material via nozzles 98, 99 and 11100. When a liquidis applied, the nozzles produce an atomized spray or mist of, whichimpacts the fibers as the fibers pass in proximity to the nozzles. Valve11102 is operated to control the flow of liquid to the respectivenozzles 1198, 1199, and 11100. After a desired quantity of additive isapplied, the valve 11102 is closed.

In some embodiments, the application zone 1190 is two to one hundredfeet long or more, e.g., 125 feet, 150 feet, 250 feet long or more,e.g., 500 feet long. Longer application zones allow for application ofover a longer period of time during passage of fibrous material throughapplication zone 1190. In some embodiments, the nozzles are spaced apartfrom about three to about four feet along the length of loop 1184.

As the fibrous material moves in loop 1184 and through the irradiatingportion of the loop 11107 that includes a horn 11109 for deliveringionizing radiation, ionizing radiation is applied to the fibrousmaterial (shielding is not shown).

As the irradiated fibrous material moves around loop 1184, it cools bythe action of gases, such as air, circulating at high speeds in the loopand it is bathed in reactive gases, such as ozone and/or oxides ofnitrogen, that are produced from the action of the ionizing radiation onthe circulating gases, such as air. After passing through theirradiating portion 11107, a cooling fluid, such as a liquid (e.g.,water) or a gas, such as liquid nitrogen at 77 K can be injected intoloop 1184 to aid in the cooling of the fibrous material. This processcan be repeated more than one time if desired, e.g., 2, 3, 4, 5, 6, 7,8, 9, 10 times or more, e.g., 15 times, to deliver the desired dose tothe fibrous material. While, as shown, the long axis of the horn isalong the direction of flow, in some implementations, the long axis ofthe horn is transverse to the direction of the flow. In someimplementations, a beam of electrons is utilized as a principal ionizingradiation source and X-rays as a secondary ionizing radiation source.X-rays can be generated by having a metal target, such as a tantalumtarget 11111, on the inside of loop 1184 such that when electrons strikethe target, X-rays are emitted.

After a desired dose is delivered to the fibrous material, the fibrousmaterial can be removed from loop 1184 via a separator 11112, which isselectively connected to loop 1184 by section 11114 and gate valve11116. When valve 11116 is opened, another valve is also opened to allowair to enter the loop 1184 to compensate for air exiting throughseparator 11112.

Referring particularly now to FIG. 11D, which shows a fluidized bedfibrous irradiating device 11121 with shielding. Fibrous material in afluid, such as a gas, such as air under pressure, is delivered to ashielded containment vessel 11123 via piping 11125 and into a shieldedfluidized bed portion 11127. Counter-current streams 11131 of fluid,such as a gas, and transverse streams 11133 of fluid, such as a gas,that is the same or different as a fluid delivered counter-currently,combine to cause turbulence in the bed portion. Ionizing radiation isapplied to the fluidized bed portion as the fibrous material is conveyedthrough the bed portion. For example, as shown, three beams of electronsfrom three Rhodotron® machines 11135, 11136 and 11137 can be utilized.

Advantageously, each beam can penetrate into the fluidized bed adifferent depth and/or each beam can emit electrons of a differentenergy, such as 1, 3, and 5 MeV. As the irradiated fibrous materialmoves through the system, it cools by the action of gases, such as air,circulating at high speeds in the system and it is bathed in reactivegases, such as ozone and/or oxides of nitrogen, that are produced fromthe action of the ionizing radiation on the circulating gases, such asair. If desired, the process can be repeated a desired number of timesuntil the fibrous material has received a desired dose. While thefluidized bed has been illustrated such that its long axis is horizontalwith the ground, in other implementations, the long axis of the bed isperpendicular to the ground so that the fibrous material falls under theinfluence of gravity.

Referring particularly now to FIG. 11E, which shows another fibrousmaterial conveying and irradiating device 11140 without shielding.Fibrous material 11144 is delivered from a bin 11142 to a first conveyer11150 at a first level above the ground and then the material istransferred to a second conveyer 11152 at a lower height than the firstconveyer. The trailing edge 11160 of the first conveyer and the leadingedge 11161 of the second conveyer 11152 defines a gap with a spacing S.For example, the spacing S can be between 4 inches and about 24 inches.Material 11144 has enough momentum to free fall under gravity and thento be captured by the second conveyer 11152 without falling into thegap. During the free fall, ionizing radiation is applied to thematerial. This arrangement can be advantageous in that the ionizingradiation is less likely to damage the conveying system because is notdirectly contacted by the radiation.

After passing through the irradiating portion, a cooling fluid, such asa liquid (e.g., water) or a gas, such as liquid nitrogen at 77 K can beapplied to the material to aid in the cooling of the fibrous material.This process can be repeated more than one time if desired, e.g., 2, 3,4, 5, 6, 7, 8, 9, 10 times or more, e.g., 15 times, to deliver thedesired dose to the fibrous material. While, as shown, the long axis ofthe horn is transverse to the direction of the material flow, other beamarrangements are possible. In some implementations, a beam of electronsis utilized as a principal ionizing radiation source and X-rays as asecondary ionizing radiation source. X-rays can be generated by having ametal target, such as a tantalum target, in the gap on the opposite sideof the material, such that as the electrons that pass through thematerial they strike the target, generating X-rays.

In one example of the use of radiation with oxidation as a pretreatment,half-gallon juice cartons made of un-printed polycoated white Kraftboard having a bulk density of 20 lb/ft³ are used as a feedstock.Cartons are folded flat and then fed into a sequence of threeshredder-shearer trains arranged in series with output from the firstshearer fed as input to the second shredder, and output from the secondshearer fed as input to the third shredder. The fibrous materialproduced by the can be sprayed with water and processed through a pelletmill operating at room temperature. The densified pellets can be placedin a glass ampoule which is sealed under an atmosphere of air. Thepellets in the ampoule are irradiated with gamma radiation for about 3hours at a dose rate of about 1 Mrad per hour to provide an irradiatedmaterial in which the cellulose has a lower molecular weight than thefibrous Kraft starting material.

Sonication

One or more sonication processing sequences can be used to treat biomassfrom a wide variety of different sources to extract useful substancesfrom the feedstock, and to provide partially degraded organic materialwhich functions as input to further processing steps and/or sequences.Sonication can reduce the recalcitrance, molecular weight, and/orcrystallinity of feedstock, such as one or more of any of the biomassmaterials described herein, e.g., one or more carbohydrate sources, suchas cellulosic or lignocellulosic materials, or starchy materials.

Referring again to FIG. 8, in one method, a first biomass material 2that includes cellulose having a first number average molecular weight(^(T)M_(N1)) is dispersed in a medium, such as water, and sonicatedand/or otherwise cavitated, to provide a second biomass material 3 thatincludes cellulose having a second number average molecular weight(^(T)M_(N2)) lower than the first number average molecular weight. Thesecond material (or the first and second material in certainembodiments) can be combined with a microorganism (e.g., a bacterium ora yeast) that can utilize the second and/or first material to produce aproduct 5.

Since the second material has cellulose having a reduced molecularweight relative to the first material, and in some instances, a reducedcrystallinity as well, the second material is generally moredispersible, swellable, and/or soluble in a solution containing themicroorganism, e.g., at a concentration of greater than 10⁶microorganisms/mL. These properties make the second material 3 moresusceptible to chemical, enzymatic, and/or microbial attack relative tothe first material 2, which can greatly improve the production rateand/or production level of a desired product, e.g., ethanol. Sonicationcan also sterilize the materials, but should not be used while themicroorganisms are supposed to be alive.

In some embodiments, the second number average molecular weight(^(T)M_(N2)) is lower than the first number average molecular weight(^(T)M_(N1)) by more than about 10 percent, e.g., 15, 20, 25, 30, 35,40, 50 percent, 60 percent, or even more than about 75 percent.

In some instances, the second material has cellulose that has acrystallinity (^(T)C₂) that is lower than the crystallinity (^(T)C₁) ofthe cellulose of the first material. For example, (^(T)C₂) can be lowerthan (^(T)C₁) by more than about 10 percent, e.g., 15, 20, 25, 30, 35,40, or even more than about 50 percent.

In some embodiments, the starting crystallinity index (prior tosonication) is from about 40 to about 87.5 percent, e.g., from about 50to about 75 percent or from about 60 to about 70 percent, and thecrystallinity index after sonication is from about 10 to about 50percent, e.g., from about 15 to about 45 percent or from about 20 toabout 40 percent. However, in certain embodiments, e.g., after extensivesonication, it is possible to have a crystallinity index of lower than 5percent. In some embodiments, the material after sonication issubstantially amorphous.

In some embodiments, the starting number average molecular weight (priorto sonication) is from about 200,000 to about 3,200,000, e.g., fromabout 250,000 to about 1,000,000 or from about 250,000 to about 700,000,and the number average molecular weight after sonication is from about50,000 to about 200,000, e.g., from about 60,000 to about 150,000 orfrom about 70,000 to about 125,000. However, in some embodiments, e.g.,after extensive sonication, it is possible to have a number averagemolecular weight of less than about 10,000 or even less than about5,000.

In some embodiments, the second material can have a level of oxidation(^(T)O₂) that is higher than the level of oxidation (^(T)O₁) of thefirst material. A higher level of oxidation of the material can aid inits dispersibility, swellability and/or solubility, further enhancingthe materials susceptibility to chemical, enzymatic or microbial attack.In some embodiments, to increase the level of the oxidation of thesecond material relative to the first material, the sonication isperformed in an oxidizing medium, producing a second material that ismore oxidized than the first material. For example, the second materialcan have more hydroxyl groups, aldehyde groups, ketone groups, estergroups or carboxylic acid groups, which can increase its hydrophilicity.

In some embodiments, the sonication medium is an aqueous medium. Ifdesired, the medium can include an oxidant, such as a peroxide (e.g.,hydrogen peroxide), a dispersing agent and/or a buffer. Examples ofdispersing agents include ionic dispersing agents, e.g., sodium laurylsulfate, and non-ionic dispersing agents, e.g., poly(ethylene glycol).

In other embodiments, the sonication medium is non-aqueous. For example,the sonication can be performed in a hydrocarbon, e.g., toluene orheptane, an ether, e.g., diethyl ether or tetrahydrofuran, or even in aliquefied gas such as argon, xenon, or nitrogen.

Without wishing to be bound by any particular theory, it is believedthat sonication breaks bonds in the cellulose by creating bubbles in themedium containing the cellulose, which grow and then violently collapse.During the collapse of the bubble, which can take place in less than ananosecond, the implosive force raises the local temperature within thebubble to about 5100 K (even higher in some instance; see, e.g., Suslicket al., Nature 434, 52-55) and generates pressures of from a few hundredatmospheres to over 1000 atmospheres or more. It is these hightemperatures and pressures that break the bonds. In addition, withoutwishing to be bound by any particular theory, it is believed thatreduced crystallinity arises, at least in part, from the extremely highcooling rates during collapse of the bubbles, which can be greater thanabout 10¹¹ K/second. The high cooling rates generally do not allow thecellulose to organize and crystallize, resulting in materials that havereduced crystallinity. Ultrasonic systems and sonochemistry arediscussed in, e.g., Olli et al., U.S. Pat. No. 5,766,764; Roberts, U.S.Pat. No. 5,828,156; Mason, Chemistry with Ultrasound, Elsevier, Oxford,(1990); Suslick (editor), Ultrasound: its Chemical, Physical andBiological Effects, VCH, Weinheim, (1988); Price, “Current Trends inSonochemistry” Royal Society of Chemistry, Cambridge, (1992); Suslick etal., Ann. Rev. Mater. Sci. 29, 295, (1999); Suslick et al., Nature 353,414 (1991); Hiller et al., Phys. Rev. Lett. 69, 1182 (1992); Barber etal., Nature, 352, 414 (1991); Suslick et al., J. Am. Chem. Soc., 108,5641 (1986); Tang et al., Chem. Comm., 2119 (2000); Wang et al.,Advanced Mater., 12, 1137 (2000); Landau et al., J. of Catalysis, 201,22 (2001); Perkas et al., Chem. Comm., 988 (2001); Nikitenko et al.,Angew. Chem. Inter. Ed. (December 2001); Shafi et al., J. Phys. Chem B103, 3358 (1999); Avivi et al., J. Amer. Chem. Soc. 121, 4196 (1999);and Avivi et al., J. Amer. Chem. Soc. 122, 4331 (2000).

Sonication Systems

FIG. 12 shows a general system in which a biomass material stream 1210is mixed with a water stream 1212 in a reservoir 1214 to form a processstream 1216. A first pump 1218 draws process stream 1216 from reservoir1214 and toward a flow cell 1224. Ultrasonic transducer 1226 transmitsultrasonic energy into process stream 1216 as the process stream flowsthrough flow cell 1224. A second pump 1230 draws process stream 1216from flow cell 1224 and toward subsequent processing.

Reservoir 1214 includes a first intake 1232 and a second intake 1234 influid communication with a volume 1236. A conveyor (not shown) deliversbiomass material stream 1210 to reservoir 1214 through first intake1232. Water stream 1212 enters reservoir 1214 through second intake1234. In some embodiments, water stream 1212 enters volume 1236 along atangent establishing a swirling flow within volume 1236. In certainembodiments, biomass material stream 1210 and water stream 1212 areintroduced into volume 1236 along opposing axes to enhance mixing withinthe volume.

Valve 1238 controls the flow of water stream 1212 through second intake1232 to produce a desired ratio of biomass material to water (e.g.,approximately 10% cellulosic material, weight by volume). For example,2000 tons/day of biomass can be combined with 1 million to 1.5 milliongallons/day, e.g., 1.25 million gallons/day, of water.

Mixing of material biomass and water in reservoir 1214 is controlled bythe size of volume 1236 and the flow rates of biomass and water into thevolume. In some embodiments, volume 1236 is sized to create a minimummixing residence time for the biomass and water. For example, when 2000tons/day of biomass and 1.25 million gallons/day of water are flowingthrough reservoir 1214, volume 1236 can be about 32,000 gallons toproduce a minimum mixing residence time of about 15 minutes.

Reservoir 1214 includes a mixer 1240 in fluid communication with volume1236. Mixer 1240 agitates the contents of volume 1236 to dispersebiomass throughout the water in the volume. For example, mixer 1240 canbe a rotating vane disposed in reservoir 1214. In some embodiments,mixer 1240 disperses the biomass substantially uniformly throughout thewater.

Reservoir 1214 further includes an exit 1242 in fluid communication withvolume 1236 and process stream 1216. The mixture of biomass and water involume 1236 flows out of reservoir 1214 via exit 1242. Exit 1242 isarranged near the bottom of reservoir 1214 to allow gravity to pull themixture of biomass and water out of reservoir 1214 and into processstream 1216.

First pump 1218 (e.g., any of several recessed impeller vortex pumpsmade by Essco Pumps & Controls, Los Angeles, Calif.) moves the contentsof process stream 1216 toward flow cell 1224. In some embodiments, firstpump 1218 agitates the contents of process stream 1216 such that themixture of cellulosic material and water is substantially uniform atinlet 1220 of flow cell 1224. For example, first pump 1218 agitatesprocess stream 1216 to create a turbulent flow that persists along theprocess stream between the first pump and inlet 1220 of flow cell 1224.

Flow cell 1224 includes a reactor volume 1244 in fluid communicationwith inlet 1220 and outlet 1222. In some embodiments, reactor volume1244 is a stainless steel tube capable of withstanding elevatedpressures (e.g., 10 bars). In addition or in the alternative, reactorvolume 1244 includes a rectangular cross section.

Flow cell 1224 further includes a heat exchanger 1246 in thermalcommunication with at least a portion of reactor volume 1244. Coolingfluid 1248 (e.g., water) flows into heat exchanger 1246 and absorbs heatgenerated when process stream 1216 is sonicated in reactor volume 1244.In some embodiments, the flow rate of cooling fluid 1248 into heatexchanger 1246 is controlled to maintain an approximately constanttemperature in reactor volume 1244. In addition or in the alternative,the temperature of cooling fluid 1248 flowing into heat exchanger 1246is controlled to maintain an approximately constant temperature inreactor volume 1244. In some embodiments, the temperature of reactorvolume 1244 is maintained at 20 to 50° C., e.g., 25, 30, 35, 40, or 45°C. Additionally or alternatively, heat transferred to cooling fluid 1248from reactor volume 1244 can be used in other parts of the overallprocess.

An adapter section 1226 creates fluid communication between reactorvolume 1244 and a booster 1250 coupled (e.g., mechanically coupled usinga flange) to ultrasonic transducer 1226. For example, adapter section1226 can include a flange and O-ring assembly arranged to create a leaktight connection between reactor volume 1244 and booster 1250. In someembodiments, ultrasonic transducer 1226 is a high-powered ultrasonictransducer made by Hielscher Ultrasonics of Teltow, Germany.

In operation, a generator 1252 delivers electricity to ultrasonictransducer 1252. Ultrasonic transducer 1226 includes a piezoelectricelement that converts the electrical energy into sound in the ultrasonicrange. In some embodiments, the materials are sonicated using soundhaving a frequency of from about 16 kHz to about 110 kHz, e.g., fromabout 18 kHz to about 75 kHz or from about 20 kHz to about 40 kHz (e.g.,sound having a frequency of 20 kHz to 40 kHz).

The ultrasonic energy is then delivered to the working medium throughbooster 1248. The ultrasonic energy traveling through booster 1248 inreactor volume 1244 creates a series of compressions and rarefactions inprocess stream 1216 with an intensity sufficient to create cavitation inprocess stream 1216. Cavitation disaggregates the cellulosic materialdispersed in process stream 1216. Cavitation also produces free radicalsin the water of process stream 1216. These free radicals act to furtherbreak down the cellulosic material in process stream 1216.

In general, 5 to 4000 MJ/m³, e.g., 10, 25, 50, 100, 250, 500, 750, 1000,2000, or 3000 MJ/m³, of ultrasonic energy is applied to process stream16 flowing at a rate of about 0.2 m³/s (about 3200 gallons/min). Afterexposure to ultrasonic energy in reactor volume 1244, process stream1216 exits flow cell 1224 through outlet 1222. Second pump 1230 movesprocess stream 1216 to subsequent processing (e.g., any of severalrecessed impeller vortex pumps made by Essco Pumps & Controls, LosAngeles, Calif.).

While certain embodiments have been described, other embodiments arepossible.

As an example, while process stream 1216 has been described as a singleflow path, other arrangements are possible. In some embodiments forexample, process stream 1216 includes multiple parallel flow paths(e.g., flowing at a rate of 10 gallon/min). In addition or in thealternative, the multiple parallel flow paths of process stream 1216flow into separate flow cells and are sonicated in parallel (e.g., usinga plurality of 16 kW ultrasonic transducers).

As another example, while a single ultrasonic transducer 1226 has beendescribed as being coupled to flow cell 1224, other arrangements arepossible. In some embodiments, a plurality of ultrasonic transducers1226 are arranged in flow cell 1224 (e.g., ten ultrasonic transducerscan be arranged in a flow cell 1224). In some embodiments, the soundwaves generated by each of the plurality of ultrasonic transducers 1226are timed (e.g., synchronized out of phase with one another) to enhancethe cavitation acting upon process stream 1216.

As another example, while a single flow cell 1224 has been described,other arrangements are possible. In some embodiments, second pump 1230moves process stream to a second flow cell where a second booster andultrasonic transducer further sonicate process stream 1216.

As still another example, while reactor volume 1244 has been describedas a closed volume, reactor volume 1244 is open to ambient conditions incertain embodiments. In such embodiments, sonication pretreatment can beperformed substantially simultaneously with other pretreatmenttechniques. For example, ultrasonic energy can be applied to processstream 1216 in reactor volume 1244 while electron beams aresimultaneously introduced into process stream 1216.

As another example, while a flow through process has been described,other arrangements are possible. In some embodiments, sonication can beperformed in a batch process. For example, a volume can be filled with a10% (weight by volume) mixture of biomass in water and exposed to soundwith intensity from about 50 W/cm² to about 600 W/cm², e.g., from about75 W/cm² to about 300 W/cm² or from about 95 W/cm² to about 200 W/cm².Additionally or alternatively, the mixture in the volume can besonicated from about 1 hour to about 24 hours, e.g., from about 1.5hours to about 12 hours, or from about 2 hours to about 10 hours. Incertain embodiments, the material is sonicated for a pre-determinedtime, and then allowed to stand for a second pre-determined time beforesonicating again.

Referring now to FIG. 13, in some embodiments, two electro-acoustictransducers are mechanically coupled to a single horn. As shown, a pairof piezoelectric transducers 60 and 62 is coupled to a slotted bar horn64 by respective intermediate coupling horns 70 and 72, the latter alsobeing known as booster horns. The mechanical vibrations provided by thetransducers, responsive to high frequency electrical energy appliedthereto, are transmitted to the respective coupling horns, which can beconstructed to provide a mechanical gain, such as a ratio of 1 to 1.2.The horns are provided with a respective mounting flange 74 and 76 forsupporting the transducer and horn assembly in a stationary housing.

The vibrations transmitted from the transducers through the coupling orbooster horns are coupled to the input surface 78 of the horn and aretransmitted through the horn to the oppositely disposed output surface80, which, during operation, is in forced engagement with a workpiece(not shown) to which the vibrations are applied.

The high frequency electrical energy provided by the power supply 82 isfed to each of the transducers, electrically connected in parallel, viaa balancing transformer 84 and a respective series connected capacitor86 and 90, one capacitor connected in series with the electricalconnection to each of the transducers. The balancing transformer isknown also as “balun” standing for “balancing unit.” The balancingtransformer includes a magnetic core 92 and a pair of identical windings94 and 96, also termed the primary winding and secondary winding,respectively.

In some embodiments, the transducers include commercially availablepiezoelectric transducers, such as Branson Ultrasonics Corporationmodels 105 or 502, each designed for operation at 20 kHz and a maximumpower rating of 3 kW. The energizing voltage for providing maximummotional excursion at the output surface of the transducer is 930 voltrms. The current flow through a transducer can vary between zero and 3.5ampere depending on the load impedance. At 930 volt rms the outputmotion is approximately 20 microns. The maximum difference in terminalvoltage for the same motional amplitude, therefore, can be 186 volt.Such a voltage difference can give rise to large circulating currentsflowing between the transducers. The balancing unit 430 assures abalanced condition by providing equal current flow through thetransducers, hence eliminating the possibility of circulating currents.The wire size of the windings must be selected for the full load currentnoted above and the maximum voltage appearing across a winding input is93 volt.

As an alternative to using ultrasonic energy, high-frequency,rotor-stator devices can be utilized. This type of device produceshigh-shear, microcavitation forces, which can disintegrate biomass incontact with such forces. Two commercially available high-frequency,rotor-stator dispersion devices are the Supraton™ devices manufacturedby Krupp Industrietechnik GmbH and marketed by Dorr-Oliver DeutschlandGmbH of Connecticut, and the Dispax™ devices manufactured and marketedby Ika-Works, Inc. of Cincinnati, Ohio. Operation of such amicrocavitation device is discussed in Stuart, U.S. Pat. No. 5,370,999.

While ultrasonic transducer 1226 has been described as including one ormore piezoelectric active elements to create ultrasonic energy, otherarrangements are possible. In some embodiments, ultrasonic transducer1226 includes active elements made of other types of magnetostrictivematerials (e.g., ferrous metals). Design and operation of such ahigh-powered ultrasonic transducer is discussed in Hansen et al., U.S.Pat. No. 6,624,539. In some embodiments, ultrasonic energy istransferred to process stream 16 through an electro-hydraulic system.

While ultrasonic transducer 1226 has been described as using theelectromagnetic response of magnetorestrictive materials to produceultrasonic energy, other arrangements are possible. In some embodiments,acoustic energy in the form of an intense shock wave can be applieddirectly to process stream 16 using an underwater spark. In someembodiments, ultrasonic energy is transferred to process stream 16through a thermo-hydraulic system. For example, acoustic waves of highenergy density can be produced by applying power across an enclosedvolume of electrolyte, thereby heating the enclosed volume and producinga pressure rise that is subsequently transmitted through a soundpropagation medium (e.g., process stream 1216). Design and operation ofsuch a thermo-hydraulic transducer is discussed in Hartmann et al., U.S.Pat. No. 6,383,152.

Pyrolysis

One or more pyrolysis treatment sequences can be used to process biomassfrom a wide variety of different sources to extract useful substancesfrom the biomass, and to provide partially degraded organic materialwhich functions as input to further processing steps and/or sequences.

Referring again to the general schematic in FIG. 8, a first biomassmaterial 2 that includes having a first number average molecular weight(^(T)M_(N1)) is pyrolyzed, e.g., by heating the first material in a tubefurnace, to provide a second material 3 that includes cellulose having asecond number average molecular weight (^(T)M_(N2)) lower than the firstnumber average molecular weight. The second material (or the first andsecond material in certain embodiments) is/are combined with amicroorganism (e.g., a bacterium or a yeast) that can utilize the secondand/or first material to produce a product 5 that.

Since the second biomass material has cellulose having a reducedmolecular weight relative to the first material, and in some instances,a reduced crystallinity as well, the second material is generally moredispersible, swellable and/or soluble in a solution containing themicroorganism, e.g., at a concentration of greater than 10⁶microorganisms/mL. These properties make the second material 3 moresusceptible to chemical, enzymatic and/or microbial attack relative tothe first material 2, which can greatly improve the production rateand/or production level of a desired product, e.g., ethanol. Pyrolysiscan also sterilize the first and second materials.

In some embodiments, the second number average molecular weight(^(T)M_(N2)) is lower than the first number average molecular weight(^(T)M_(N1)) by more than about 10 percent, e.g., 15, 20, 25, 30, 35,40, 50 percent, 60 percent, or even more than about 75 percent.

In some instances, the second material has cellulose that has acrystallinity (^(T)C₂) that is lower than the crystallinity (^(T)C₁) ofthe cellulose of the first material. For example, (^(T)C₂) can be lowerthan (^(T)C₁) by more than about 10 percent, e.g., 15, 20, 25, 30, 35,40, or even more than about 50 percent.

In some embodiments, the starting crystallinity (prior to pyrolysis) isfrom about 40 to about 87.5 percent, e.g., from about 50 to about 75percent or from about 60 to about 70 percent, and the crystallinityindex after pyrolysis is from about 10 to about 50 percent, e.g., fromabout 15 to about 45 percent or from about 20 to about 40 percent.However, in certain embodiments, e.g., after extensive pyrolysis, it ispossible to have a crystallinity index of lower than 5 percent. In someembodiments, the material after pyrolysis is substantially amorphous.

In some embodiments, the starting number average molecular weight (priorto pyrolysis) is from about 200,000 to about 3,200,000, e.g., from about250,000 to about 1,000,000 or from about 250,000 to about 700,000, andthe number average molecular weight after pyrolysis is from about 50,000to about 200,000, e.g., from about 60,000 to about 150,000 or from about70,000 to about 125,000. However, in some embodiments, e.g., afterextensive pyrolysis, it is possible to have a number average molecularweight of less than about 10,000 or even less than about 5,000.

In some embodiments, the second material can have a level of oxidation(^(T)O₂) that is higher than the level of oxidation (^(T)O₁) of thefirst material. A higher level of oxidation of the material can aid inits dispersibility, swellability and/or solubility, further enhancingthe materials susceptibility to chemical, enzymatic or microbial attack.In some embodiments, to increase the level of the oxidation of thesecond material relative to the first material, the pyrolysis isperformed in an oxidizing environment, producing a second material thatis more oxidized than the first material. For example, the secondmaterial can have more hydroxyl groups, aldehyde groups, ketone groups,ester groups or carboxylic acid groups, which can increase itshydrophilicity.

In some embodiments, the pyrolysis of the materials is continuous. Inother embodiments, the material is pyrolyzed for a pre-determined time,and then allowed to cool for a second pre-determined time beforepyrolyzing again.

Pyrolysis Systems

FIG. 14 shows a process flow diagram 6000 that includes various steps ina pyrolytic feedstock pretreatment system. In first step 6010, a supplyof dry feedstock is received from a feed source.

As described above, the dry biomass from the feed source can bepre-processed prior to delivery to the pyrolysis chamber. For example,if the biomass is derived from plant sources, certain portions of theplant material can be removed prior to collection of the plant materialand/or before the plant material is delivered by the feedstock transportdevice. Alternatively, or in addition, the biomass feedstock can besubjected to mechanical processing 6020 (e.g., to reduce the averagelength of fibers in the feedstock) prior to delivery to the pyrolysischamber.

Following mechanical processing, the biomass undergoes a moistureadjustment step 6030. The nature of the moisture adjustment step dependsupon the moisture content of the mechanically processed biomass.Typically, pyrolysis of biomass occurs most efficiently when themoisture content of the feedstock is between about 10% and about 30%(e.g., between 15% and 25%) by weight of the feedstock. If the moisturecontent of the feedstock is larger than about 40% by weight, the extrathermal load presented by the water content of the biomass increases theenergy consumption of subsequent pyrolysis steps.

In some embodiments, if the biomass has a moisture content which islarger than about 30% by weight, drier biomass material 6220, which hasa low moisture content, can be blended in, creating a feedstock mixturein step 6030 with an average moisture content that is within the limitsdiscussed above. In certain embodiments, biomass with a high moisturecontent can simply be dried by dispersing the biomass material on amoving conveyor that cycles the biomass through an in-line heating unit.The heating unit evaporates a portion of the water present in thefeedstock.

In some embodiments, if the biomass from step 6020 has a moisturecontent which is too low (e.g., lower than about 10% by weight), themechanically processed biomass can be combined with wetter feedstockmaterial 6230 with a higher moisture content, such as sewage sludge.Alternatively, or in addition, water 6240 can be added to the drybiomass from step 6020 to increase its moisture content.

In step 6040, the biomass—now with its moisture content adjusted to fallwithin suitable limits—can be preheated in an optional preheating step6040. Treatment step 6040 can be used to increase the temperature of thebiomass to between 75° C. and 150° C. in preparation for subsequentpyrolysis of the biomass. Depending upon the nature of the biomass andthe particular design of the pyrolysis chamber, preheating the biomasscan ensure that heat distribution within the biomass feedstock remainsmore uniform during pyrolysis, and can reduce the thermal load on thepyrolysis chamber.

The feedstock is then transported to a pyrolysis chamber to undergopyrolysis in step 6050. In some embodiments, transport of the feedstockis assisted by adding one or more pressurized gases 6210 to thefeedstock stream. The gases create a pressure gradient in a feedstocktransport conduit, propelling the feedstock into the pyrolysis chamber(and even through the pyrolysis chamber). In certain embodiments,transport of the feedstock occurs mechanically; that is, a transportsystem that includes a conveyor such as an auger transports thefeedstock to the pyrolysis chamber.

Other gases 6210 can also be added to the feedstock prior to thepyrolysis chamber. In some embodiments, for example, one or morecatalyst gases can be added to the feedstock to assist decomposition ofthe feedstock during pyrolysis. In certain embodiments, one or morescavenging agents can be added to the feedstock to trap volatilematerials released during pyrolysis. For example, various sulfur-basedcompounds such as sulfides can be liberated during pyrolysis, and anagent such as hydrogen gas can be added to the feedstock to causedesulfurization of the pyrolysis products. Hydrogen combines withsulfides to form hydrogen sulfide gas, which can be removed from thepyrolyzed feedstock.

Pyrolysis of the feedstock within the chamber can include heating thefeedstock to relatively high temperatures to cause partial decompositionof the feedstock. Typically, the feedstock is heated to a temperature ina range from 150° C. to 1100° C. The temperature to which the feedstockis heated depends upon a number of factors, including the composition ofthe feedstock, the feedstock average particle size, the moisturecontent, and the desired pyrolysis products. For many types of biomassfeedstock, for example, pyrolysis temperatures between 300° C. and 550°C. are used.

The residence time of the feedstock within the pyrolysis chambergenerally depends upon a number of factors, including the pyrolysistemperature, the composition of the feedstock, the feedstock averageparticle size, the moisture content, and the desired pyrolysis products.In some embodiments, feedstock materials are pyrolyzed at a temperaturejust above the decomposition temperature for the material in an inertatmosphere, e.g., from about 2° C. above to about 10° C. above thedecomposition temperature or from about 3° C. above to about 7° C. abovethe decomposition temperature. In such embodiments, the material isgenerally kept at this temperature for greater than 0.5 hours, e.g.,greater than 1.0 hours or greater than about 2.0 hours. In otherembodiments, the materials are pyrolyzed at a temperature well above thedecomposition temperature for the material in an inert atmosphere, e.g.,from about 75° C. above to about 175° C. above the decompositiontemperature or from about 85° C. above to about 150° C. above thedecomposition temperature. In such embodiments, the material isgenerally kept at this temperature for less than 0.5 hour, e.g., less 20minutes, less than 10 minutes, less than 5 minutes or less than 2minutes. In still other embodiments, the materials are pyrolyzed at anextreme temperature, e.g., from about 200° C. above to about 500° C.above the decomposition temperature of the material in an inertenvironment or from about 250° C. above to about 400° C. above thedecomposition temperature. In such embodiments, the material usgenerally kept at this temperature for less than 1 minute, e.g., lessthan 30 seconds, less than 15 seconds, less than 10 seconds, less than 5seconds, less than 1 second or less than 500 ms. Such embodiments aretypically referred to as flash pyrolysis.

In some embodiments, the feedstock is heated relatively rapidly to theselected pyrolysis temperature within the chamber. For example, thechamber can be designed to heat the feedstock at a rate of between 500°C./s and 11,000° C./s. Typical heating rates for biomass-derivedfeedstock material are from 500° C./s to 1000° C./s, for example.

A turbulent flow of feedstock material within the pyrolysis chamber isusually advantageous, as it ensures relatively efficient heat transferto the feedstock material from the heating sub-system. Turbulent flowcan be achieved by blowing the feedstock material through the chamberusing one or more injected carrier gases 6210, for example. In general,the carrier gases are relatively inert towards the feedstock material,even at the high temperatures in the pyrolysis chamber. Exemplarycarrier gases include, for example, nitrogen, argon, methane, carbonmonoxide, and carbon dioxide. Alternatively, or in addition, mechanicaltransport systems such as augers can transport and circulate thefeedstock within the pyrolysis chamber to create a turbulent feedstockflow.

In some embodiments, pyrolysis of the feedstock occurs substantially inthe absence of oxygen and other reactive gases. Oxygen can be removedfrom the pyrolysis chamber by periodic purging of the chamber with highpressure nitrogen (e.g., at nitrogen pressures of 2 bar or more).Following purging of the chamber, a gas mixture present in the pyrolysischamber (e.g., during pyrolysis of the feedstock) can include less than4 mole % oxygen (e.g., less than 1 mole % oxygen, and even less than 0.5mole % oxygen). The absence of oxygen ensures that ignition of thefeedstock does not occur at the elevated pyrolysis temperatures.

In certain embodiments, relatively small amounts of oxygen can beintroduced into the feedstock and are present during pyrolysis. Thistechnique is referred to as oxidative pyrolysis. Typically, oxidativepyrolysis occurs in multiple heating stages. For example, in a firstheating stage, the feedstock is heated in the presence of oxygen tocause partial oxidation of the feedstock. This stage consumes theavailable oxygen in the pyrolysis chamber. Then, in subsequent heatingstages, the feedstock temperature is further elevated. With all of theoxygen in the chamber consumed, however, feedstock combustion does notoccur, and combustion-free pyrolytic decomposition of the feedstock(e.g., to generate hydrocarbon products) occurs. In general, the processof heating feedstock in the pyrolysis chamber to initiate decompositionis endothermic. However, in oxidative pyrolysis, formation of carbondioxide by oxidation of the feedstock is an exothermic process. The heatreleased from carbon dioxide formation can assist further pyrolysisheating stages, thereby lessening the thermal load presented by thefeedstock.

In some embodiments, pyrolysis occurs in an inert environment, such aswhile feedstock materials are bathed in argon or nitrogen gas. Incertain embodiments, pyrolysis can occur in an oxidizing environment,such as in air or argon enriched in air. In some embodiments, pyrolysiscan take place in a reducing environment, such as while feedstockmaterials are bathed in hydrogen gas. To aid pyrolysis, various chemicalagents, such as oxidants, reductants, acids or bases can be added to thematerial prior to or during pyrolysis. For example, sulfuric acid can beadded, or a peroxide (e.g., benzoyl peroxide) can be added.

As discussed above, a variety of different processing conditions can beused, depending upon factors such as the feedstock composition and thedesired pyrolysis products. For example, for cellulose-containingfeedstock material, relatively mild pyrolysis conditions can beemployed, including flash pyrolysis temperatures between 375° C. and450° C., and residence times of less than 1 second. As another example,for organic solid waste material such as sewage sludge, flash pyrolysistemperatures between 500° C. and 650° C. are typically used, withresidence times of between 0.5 and 3 seconds. In general, many of thepyrolysis process parameters, including residence time, pyrolysistemperature, feedstock turbulence, moisture content, feedstockcomposition, pyrolysis product composition, and additive gas compositioncan be regulated automatically by a system of regulators and anautomated control system.

Following pyrolysis step 6050, the pyrolysis products undergo aquenching step 6250 to reduce the temperature of the products prior tofurther processing. Typically, quenching step 6250 includes spraying thepyrolysis products with streams of cooling water 6260. The cooling wateralso forms a slurry that includes solid, undissolved product materialand various dissolved products. Also present in the product stream is amixture that includes various gases, including product gases, carriergases, and other types of process gases.

The product stream is transported via in-line piping to a gas separatorthat performs a gas separation step 6060, in which product gases andother gases are separated from the slurry formed by quenching thepyrolysis products. The separated gas mixture is optionally directed toa blower 6130, which increases the gas pressure by blowing air into themixture. The gas mixture can be subjected to a filtration step 6140, inwhich the gas mixture passes through one or more filters (e.g.,activated charcoal filters) to remove particulates and other impurities.In a subsequent step 6150, the filtered gas can be compressed and storedfor further use. Alternatively, the filtered gas can be subjected tofurther processing steps 6160. For example, in some embodiments, thefiltered gas can be condensed to separate different gaseous compoundswithin the gas mixture. The different compounds can include, forexample, various hydrocarbon products (e.g., alcohols, alkanes, alkenes,alkynes, ethers) produced during pyrolysis. In certain embodiments, thefiltered gas containing a mixture of hydrocarbon components can becombined with steam gas 6170 (e.g., a mixture of water vapor and oxygen)and subjected to a cracking process to reduce molecular weights of thehydrocarbon components.

In some embodiments, the pyrolysis chamber includes heat sources thatburn hydrocarbon gases such as methane, propane, and/or butane to heatthe feedstock. A portion 6270 of the separated gases can be recirculatedinto the pyrolysis chamber for combustion, to generate process heat tosustain the pyrolysis process.

In certain embodiments, the pyrolysis chamber can receive process heatthat can be used to increase the temperature of feedstock materials. Forexample, irradiating feedstock with radiation (e.g., gamma radiation,electron beam radiation, or other types of radiation) can heat thefeedstock materials to relatively high temperatures. The heatedfeedstock materials can be cooled by a heat exchange system that removessome of the excess heat from the irradiated feedstock. The heat exchangesystem can be configured to transport some of the heat energy to thepyrolysis chamber to heat (or pre-heat) feedstock material, therebyreducing energy cost for the pyrolysis process.

The slurry containing liquid and solid pyrolysis products can undergo anoptional de-watering step 6070, in which excess water can be removedfrom the slurry via processes such as mechanical pressing andevaporation. The excess water 6280 can be filtered and then recirculatedfor further use in quenching the pyrolysis decomposition products instep 6250.

The de-watered slurry then undergoes a mechanical separation step 6080,in which solid product material 6110 is separated from liquid productmaterial 6090 by a series of increasingly fine filters. In step 6100,the liquid product material 6090 can then be condensed (e.g., viaevaporation) to remove waste water 6190, and purified by processes suchas extraction. Extraction can include the addition of one or moreorganic solvents 6180, for example, to separate products such as oilsfrom products such as alcohols. Suitable organic solvents include, forexample, various hydrocarbons and halo-hydrocarbons. The purified liquidproducts 6200 can then be subjected to further processing steps. Wastewater 6190 can be filtered if necessary, and recirculated for furtheruse in quenching the pyrolysis decomposition products in step 6250.

After separation in step 6080, the solid product material 6110 isoptionally subjected to a drying step 6120 that can include evaporationof water. Solid material 6110 can then be stored for later use, orsubjected to further processing steps, as appropriate.

The pyrolysis process parameters discussed above are exemplary. Ingeneral, values of these parameters can vary widely according to thenature of the feedstock and the desired products. Moreover, a widevariety of different pyrolysis techniques, including using heat sourcessuch as hydrocarbon flames and/or furnaces, infrared lasers, microwaveheaters, induction heaters, resistive heaters, and other heating devicesand configurations can be used.

A wide variety of different pyrolysis chambers can be used to decomposethe feedstock. In some embodiments, for example, pyrolyzing feedstockcan include heating the material using a resistive heating member, suchas a metal filament or metal ribbon. The heating can occur by directcontact between the resistive heating member and the material.

In certain embodiments, pyrolyzing can include heating the material byinduction, such as by using a Currie-Point pyrolyzer. In someembodiments, pyrolyzing can include heating the material by theapplication of radiation, such as infrared radiation. The radiation canbe generated by a laser, such as an infrared laser.

In certain embodiments, pyrolyzing can include heating the material witha convective heat. The convective heat can be generated by a flowingstream of heated gas. The heated gas can be maintained at a temperatureof less than about 1200° C., such as less than 1000° C., less than 750°C., less than 600° C., less than 400° C. or even less than 300° C. Theheated gas can be maintained at a temperature of greater than about 250°C. The convective heat can be generated by a hot body surrounding thefirst material, such as in a furnace.

In some embodiments, pyrolyzing can include heating the material withsteam at a temperature above about 250° C.

An embodiment of a pyrolysis chamber is shown in FIG. 15. Chamber 6500includes an insulated chamber wall 6510 with a vent 6600 for exhaustgases, a plurality of burners 6520 that generate heat for the pyrolysisprocess, a transport duct 6530 for transporting the feedstock throughchamber 6500, augers 6590 for moving the feedstock through duct 6530 ina turbulent flow, and a quenching system 6540 that includes an auger6610 for moving the pyrolysis products, water jets 6550 for spraying thepyrolysis products with cooling water, and a gas separator forseparating gaseous products 6580 from a slurry 6570 containing solid andliquid products.

Another embodiment of a pyrolysis chamber is shown in FIG. 16. Chamber6700 includes an insulated chamber wall 6710, a feedstock supply duct6720, a sloped inner chamber wall 6730, burners 6740 that generate heatfor the pyrolysis process, a vent 6750 for exhaust gases, and a gasseparator 6760 for separating gaseous products 6770 from liquid andsolid products 6780. Chamber 6700 is configured to rotate in thedirection shown by arrow 6790 to ensure adequate mixing and turbulentflow of the feedstock within the chamber.

A further embodiment of a pyrolysis chamber is shown in FIG. 17.Filament pyrolyzer 1712 includes a sample holder 1713 with resistiveheating element 1714 in the form of a wire winding through the openspace defined by the sample holder 1713. Optionally, the heated elementcan be spun about axis 1715 (as indicated by arrow 1716) to tumble thematerial that includes the cellulosic material in sample holder 1713.The space 1718 defined by enclosure 1719 is maintained at a temperatureabove room temperature, e.g., 200 to 250° C. In a typical usage, acarrier gas, e.g., an inert gas, or an oxidizing or reducing gas,traverses through the sample holder 1713 while the resistive heatingelement is rotated and heated to a desired temperature, e.g., 325° C.After an appropriate time, e.g., 5 to 10 minutes, the pyrolyzed materialis emptied from the sample holder. The system shown in FIG. 17 can bescaled and made continuous. For example, rather than a wire as theheating member, the heating member can be an auger screw. Material cancontinuously fall into the sample holder, striking a heated screw thatpyrolizes the material. At the same time, the screw can push thepyrolyzed material out of the sample holder to allow for the entry offresh, unpyrolyzed material.

Another embodiment of a pyrolysis chamber is shown in FIG. 18, whichfeatures a Curie-Point pyrolyzer 1820 that includes a sample chamber1821 housing a ferromagnetic foil 1822. Surrounding the sample chamber1821 is an RF coil 1823. The space 1824 defined by enclosure 1825 ismaintained at a temperature above room temperature, e.g., 200 to 250° C.In a typical usage, a carrier gas traverses through the sample chamber1821 while the foil 1822 is inductively heated by an applied RF field topyrolize the material at a desired temperature.

Yet another embodiment of a pyrolysis chamber is shown in FIG. 19.Furnace pyrolyzer 130 includes a movable sample holder 131 and a furnace132. In a typical usage, the sample is lowered (as indicated by arrow137) into a hot zone 135 of furnace 132, while a carrier gas fills thehousing 136 and traverses through the sample holder 131. The sample isheated to the desired temperature for a desired time to provide apyrolyzed product. The pyrolyzed product is removed from the pyrolyzerby raising the sample holder (as indicated by arrow 134).

In certain embodiments, as shown in FIG. 20, a cellulosic target 140 canbe pyrolyzed by treating the target, which is housed in a vacuum chamber141, with laser light, e.g., light having a wavelength of from about 225nm to about 1500 nm. For example, the target can be ablated at 266 nm,using the fourth harmonic of a Nd-YAG laser (Spectra Physics, GCR170,San Jose, Calif.). The optical configuration shown allows the nearlymonochromatic light 143 generated by the laser 142 to be directed usingmirrors 144 and 145 onto the target after passing though a lens 146 inthe vacuum chamber 141. Typically, the pressure in the vacuum chamber ismaintained at less than about 10⁻⁶ mm Hg. In some embodiments, infraredradiation is used, e.g., 1.06 micron radiation from a Nd-YAG laser. Insuch embodiments, a infrared sensitive dye can be combined with thecellulosic material to produce a cellulosic target. The infrared dye canenhance the heating of the cellulosic material. Laser ablation isdescribed by Blanchet-Fincher et al. in U.S. Pat. No. 5,942,649.

Referring to FIG. 21, in some embodiments, a cellulosic material can beflash pyrolyzed by coating a tungsten filament 150, such as a 5 to 25mil tungsten filament, with the desired cellulosic material while thematerial is housed in a vacuum chamber 151. To affect pyrolysis, currentis passed through the filament, which causes a rapid heating of thefilament for a desired time. Typically, the heating is continued forseconds before allowing the filament to cool. In some embodiments, theheating is performed a number of times to effect the desired amount ofpyrolysis.

In certain embodiments, carbohydrate-containing biomass material can beheated in an absence of oxygen in a fluidized bed reactor. If desired,the carbohydrate containing biomass can have relatively thincross-sections, and can include any of the fibrous materials describedherein, for efficient heat transfer. The material can be heated bythermal transfer from a hot metal or ceramic, such as glass beads orsand in the reactor, and the resulting pyrolysis liquid or oil can betransported to a central production plant to manufacture a product.

Oxidation

One or more oxidative processing sequences can be used to process rawbiomass feedstock from a wide variety of different sources to extractuseful substances from the feedstock, and to provide partially degradedorganic material which functions as input to further processing stepsand/or sequences.

Referring again to FIG. 8, a first biomass material 2 that includescellulose having a first number average molecular weight (^(T)M_(N1))and having a first oxygen content (^(T)O₁) is oxidized, e.g., by heatingthe first material in a tube furnace in stream of air or oxygen-enrichedair, to provide a second material 3 that includes cellulose having asecond number average molecular weight (^(T)M_(N2)) and having a secondoxygen content (^(T)O₂) higher than the first oxygen content (^(T)O₁).The second material (or the first and second material in certainembodiments) can be, e.g., combined with a material, such as amicroorganism, to provide a composite 4, or another product 5. Providinga higher level of oxidation can improve dispersibility of the oxidizedmaterial, e.g., in a solvent.

Such materials can also be combined with a solid and/or a liquid. Forexample, the liquid can be in the form of a solution and the solid canbe particulate in form. The liquid and/or solid can include amicroorganism, e.g., a bacterium, and/or an enzyme. For example, thebacterium and/or enzyme can work on the cellulosic or lignocellulosicmaterial to produce a product, such as a protein. Exemplary products aredescribed in FIBROUS MATERIALS AND COMPOSITES,” U.S. Ser. No.11/453,951, filed Jun. 15, 2006.

In some embodiments, the second number average molecular weight is notmore 97 percent lower than the first number average molecular weight,e.g., not more than 95 percent, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45,40, 30, 20, 12.5, 10.0, 7.5, 5.0, 4.0, 3.0, 2.5, 2.0 or not more than1.0 percent lower than the first number average molecular weight. Theamount of reduction of molecular weight will depend upon theapplication.

For example, in some embodiments the starting number average molecularweight (prior to oxidation) is from about 200,000 to about 3,200,000,e.g., from about 250,000 to about 1,000,000 or from about 250,000 toabout 700,000, and the number average molecular weight after oxidationis from about 175,000 to about 3,000,000, e.g., from about 200,000 toabout 750,000 or from about 225,000 to about 600,000.

Resins utilized can be thermosets or thermoplastics. Examples ofthermoplastic resins include rigid and elastomeric thermoplastics. Rigidthermoplastics include polyolefins (e.g., polyethylene, polypropylene,or polyolefin copolymers), polyesters (e.g., polyethyleneterephthalate), polyamides (e.g., nylon 6, 6/12 or 6/10), andpolyethyleneimines. Examples of elastomeric thermoplastic resins includeelastomeric styrenic copolymers (e.g., styrene-ethylene-butylene-styrenecopolymers), polyamide elastomers (e.g., polyether-polyamide copolymers)and ethylene-vinyl acetate copolymer.

In particular embodiments, lignin is utilized, e.g., any lignin that isgenerated in any process described herein.

In some embodiments, the thermoplastic resin has a melt flow rate ofbetween 10 g/10 minutes to 60 g/10 minutes, e.g., between 20 g/10minutes to 50 g/10 minutes, or between 30 g/10 minutes to 45 g/10minutes, as measured using ASTM 1238. In certain embodiments, compatibleblends of any of the above thermoplastic resins can be used.

In some embodiments, the thermoplastic resin has a polydispersity index(PDI), e.g., a ratio of the weight average molecular weight to thenumber average molecular weight, of greater than 1.5, e.g., greater than2.0, greater than 2.5, greater than 5.0, greater than 7.5, or evengreater than 10.0.

In specific embodiments, polyolefins or blends of polyolefins areutilized as the thermoplastic resin.

Examples of thermosetting resins include natural rubber,butadiene-rubber and polyurethanes.

In some embodiments the starting number average molecular weight (priorto oxidation) is from about 200,000 to about 3,200,000, e.g., from about250,000 to about 1,000,000 or from about 250,000 to about 700,000, andthe number average molecular weight after oxidation is from about 50,000to about 200,000, e.g., from about 60,000 to about 150,000 or from about70,000 to about 125,000. However, in some embodiments, e.g., afterextensive oxidation, it is possible to have a number average molecularweight of less than about 10,000 or even less than about 5,000.

In some embodiments, the second oxygen content is at least about fivepercent higher than the first oxygen content, e.g., 7.5 percent higher,10.0 percent higher, 12.5 percent higher, 15.0 percent higher or 17.5percent higher. In some preferred embodiments, the second oxygen contentis at least about 20.0 percent higher than the oxygen content of thefirst material. Oxygen content is measured by elemental analysis bypyrolyzing a sample in a furnace operating 1300° C. or higher. Asuitable elemental analyzer is the LECO CHNS-932 analyzer with a VTF-900high temperature pyrolysis furnace.

In some embodiments, oxidation of first material 200 does not result ina substantial change in the crystallinity of the cellulose. However, insome instances, e.g., after extreme oxidation, the second material hascellulose that has as crystallinity (^(T)C₂) that is lower than thecrystallinity (^(T)C₁) of the cellulose of the first material. Forexample, (^(T)C₂) can be lower than (^(T)C₁) by more than about 5percent, e.g., 10, 15, 20, or even 25 percent. This can be desirable toenhance solubility of the materials in a liquid, such as a liquid thatincludes a bacterium and/or an enzyme.

In some embodiments, the starting crystallinity index (prior tooxidation) is from about 40 to about 87.5 percent, e.g., from about 50to about 75 percent or from about 60 to about 70 percent, and thecrystallinity index after oxidation is from about 30 to about 75.0percent, e.g., from about 35.0 to about 70.0 percent or from about 37.5to about 65.0 percent. However, in certain embodiments, e.g., afterextensive oxidation, it is possible to have a crystallinity index oflower than 5 percent. In some embodiments, the material after oxidationis substantially amorphous.

Without wishing to be bound by any particular theory, it is believedthat oxidation increases the number of hydrogen-bonding groups on thecellulose, such as hydroxyl groups, aldehyde groups, ketone groupscarboxylic acid groups or anhydride groups, which can increase itsdispersibility and/or its solubility (e.g., in a liquid). To furtherimprove dispersibility in a resin, the resin can include a componentthat includes hydrogen-bonding groups, such as one or more anhydridegroups, carboxylic acid groups, hydroxyl groups, amide groups, aminegroups or mixtures of any of these groups. In some preferredembodiments, the component includes a polymer copolymerized with and/orgrafted with maleic anhydride. Such materials are available from DuPontunder the trade name FUSABOND®.

Generally, oxidation of first material 200 occurs in an oxidizingenvironment. For example, the oxidation can be effected or aided bypyrolysis in an oxidizing environment, such as in air or argon enrichedin air. To aid in the oxidation, various chemical agents, such asoxidants, acids or bases can be added to the material prior to or duringoxidation. For example, a peroxide (e.g., benzoyl peroxide) can be addedprior to oxidation.

Oxidation Systems

FIG. 22 shows a process flow diagram 5000 that includes various steps inan oxidative feedstock pretreatment system. In first step 5010, a supplyof dry feedstock is received from a feed source. The feed source caninclude, for example, a storage bed or container that is connected to anin-line oxidation reactor via a conveyor belt or another feedstocktransport device.

As described above, the dry feedstock from the feed source can bepretreated prior to delivery to the oxidation reactor. For example, ifthe feedstock is derived from plant sources, certain portions of theplant material can be removed prior to collection of the plant materialand/or before the plant material is delivered by the feedstock transportdevice. Alternatively, or in addition, the biomass feedstock can besubjected to mechanical processing (e.g., to reduce the average lengthof fibers in the feedstock) prior to delivery to the oxidation reactor.

Following mechanical processing 5020, feedstock 5030 is transported to amixing system which introduces water 5150 into the feedstock in amechanical mixing process. Combining water with the processed feedstockin mixing step 5040 creates an aqueous feedstock slurry 5050, which canthen be treated with one or more oxidizing agents.

Typically, one liter of water is added to the mixture for every 0.02 kgto 1.0 kg of dry feedstock. The ratio of feedstock to water in themixture depends upon the source of the feedstock and the specificoxidizing agents used further downstream in the overall process. Forexample, in typical industrial processing sequences for lignocellulosicbiomass, aqueous feedstock slurry 5050 includes from about 0.5 kg toabout 1.0 kg of dry biomass per liter of water.

In some embodiments, one or more fiber-protecting additives 5170 canalso be added to the feedstock slurry in feedstock mixing step 5040.Fiber-protecting additives help to reduce degradation of certain typesof biomass fibers (e.g., cellulose fibers) during oxidation of thefeedstock. Fiber-protecting additives can be used, for example, if adesired product from processing a lignocellulosic feedstock includescellulose fibers. Exemplary fiber-protecting additives include magnesiumcompounds such as magnesium hydroxide. Concentrations offiber-protecting additives in feedstock slurry 5050 can be from 0.1% to0.4% of the dry weight of the biomass feedstock, for example.

In certain embodiments, aqueous feedstock slurry 5050 can be subjectedto an optional extraction 5180 with an organic solvent to removewater-insoluble substances from the slurry. For example, extraction ofslurry 5050 with one or more organic solvents yields a purified slurryand an organic waste stream 5210 that includes water-insoluble materialssuch as fats, oils, and other non-polar, hydrocarbon-based substances.Suitable solvents for performing extraction of slurry 5050 includevarious alcohols, hydrocarbons, and halo-hydrocarbons, for example.

In some embodiments, aqueous feedstock slurry 5050 can be subjected toan optional thermal treatment 5190 to further prepare the feedstock foroxidation. An example of a thermal treatment includes heating thefeedstock slurry in the presence of pressurized steam. In fibrousbiomass feedstock, the pressurized steam swells the fibers, exposing alarger fraction of fiber surfaces to the aqueous solvent and tooxidizing agents that are introduced in subsequent processing steps.

In certain embodiments, aqueous feedstock slurry 5050 can be subjectedto an optional treatment with basic agents 5200. Treatment with one ormore basic agents can help to separate lignin from cellulose inlignocellulosic biomass feedstock, thereby improving subsequentoxidation of the feedstock. Exemplary basic agents include alkali andalkaline earth hydroxides such as sodium hydroxide, potassium hydroxide,and calcium hydroxide. In general, a variety of basic agents can beused, typically in concentrations from about 0.01% to about 0.5% of thedry weight of the feedstock.

Aqueous feedstock slurry 5050 is transported (e.g., by an in-line pipingsystem) to a chamber, which can be an oxidation preprocessing chamber oran oxidation reactor. In oxidation preprocessing step 5060, one or moreoxidizing agents 5160 are added to feedstock slurry 5050 to form anoxidizing medium. In some embodiments, for example, oxidizing agents5160 can include hydrogen peroxide. Hydrogen peroxide can be added toslurry 5050 as an aqueous solution, and in proportions ranging from 3%to between 30% and 35% by weight of slurry 5050. Hydrogen peroxide has anumber of advantages as an oxidizing agent. For example, aqueoushydrogen peroxide solution is relatively inexpensive, is relativelychemically stable, is not particularly hazardous relative to otheroxidizing agents (and therefore does not require burdensome handlingprocedures and expensive safety equipment). Moreover, hydrogen peroxidedecomposes to form water during oxidation of feedstock, so that wastestream cleanup is relatively straightforward and inexpensive.

In certain embodiments, oxidizing agents 5160 can include oxygen (e.g.,oxygen gas) either alone, or in combination with hydrogen peroxide.Oxygen gas can be bubbled into slurry 5050 in proportions ranging from0.5% to 10% by weight of slurry 5050. Alternatively, or in addition,oxygen gas can also be introduced into a gaseous phase in equilibriumwith slurry 5050 (e.g., a vapor head above slurry 5050). The oxygen gascan be introduced into either an oxidation preprocessing chamber or intoan oxidation reactor (or into both), depending upon the configuration ofthe oxidative processing system. Typically, for example, the partialpressure of oxygen in the vapor above slurry 5050 is larger than theambient pressure of oxygen, and ranges from 0.5 bar to 35 bar, dependingupon the nature of the feedstock.

The oxygen gas can be introduced in pure form, or can be mixed with oneor more carrier gases. For example, in some embodiments, high-pressureair provides the oxygen in the vapor. In certain embodiments, oxygen gascan be supplied continuously to the vapor phase to ensure that aconcentration of oxygen in the vapor remains within certainpredetermined limits during processing of the feedstock. In someembodiments, oxygen gas can be introduced initially in sufficientconcentration to oxidize the feedstock, and then the feedstock can betransported to a closed, pressurized vessel (e.g., an oxidation reactor)for processing.

In certain embodiments, oxidizing agents 5160 can include nascent oxygen(e.g., oxygen radicals). Typically, nascent oxygen is produced as neededin an oxidation reactor or in a chamber in fluid communication with anoxidation reactor by one or more decomposition reactions. For example,in some embodiments, nascent oxygen can be produced from a reactionbetween NO and O₂ in a gas mixture or in solution. In certainembodiments, nascent oxygen can be produced from decomposition of HOClin solution. Other methods by which nascent oxygen can be producedinclude via electrochemical generation in electrolyte solution, forexample.

In general, nascent oxygen is an efficient oxidizing agent due to therelatively high reactivity of the oxygen radical. However, nascentoxygen can also be a relatively selective oxidizing agent. For example,when lignocellulosic feedstock is treated with nascent oxygen, selectiveoxidation of lignin occurs in preference to the other components of thefeedstock such as cellulose. As a result, oxidation of feedstock withnascent oxygen provides a method for selective removal of the ligninfraction in certain feedstocks. Typically, nascent oxygen concentrationsof between about 0.5% and 5% of the dry weight of the feedstock are usedto effect efficient oxidation.

Without wishing to be bound by theory, it is believed that nascentoxygen reacts with lignocellulosic feedstock according to at least twodifferent mechanisms. In a first mechanism, nascent oxygen undergoes anaddition reaction with the lignin, resulting in partial oxidation of thelignin, which solubilizes the lignin in aqueous solution. As a result,the solubilized lignin can be removed from the rest of the feedstock viawashing. In a second mechanism, nascent oxygen disrupts butanecross-links and/or opens aromatic rings that are connected via thebutane cross-links. As a result, solubility of the lignin in aqueoussolution increases, and the lignin fraction can be separated from theremainder of the feedstock via washing.

In some embodiments, oxidizing agents 5160 include ozone (O₃). The useof ozone can introduce several chemical-handling considerations in theoxidation processing sequence. If heated too vigorously, an aqueoussolution of ozone can decompose violently, with potentially adverseconsequences for both human system operators and system equipment.Accordingly, ozone is typically generated in a thermally isolated,thick-walled vessel separate from the vessel that contains the feedstockslurry, and transported thereto at the appropriate process stage.

Without wishing to be bound by theory, it is believed that ozonedecomposes into oxygen and oxygen radicals, and that the oxygen radicals(e.g., nascent oxygen) are responsible for the oxidizing properties ofozone in the manner discussed above. Ozone typically preferentiallyoxidizes the lignin fraction in lignocellulosic materials, leaving thecellulose fraction relatively undisturbed.

Conditions for ozone-based oxidation of biomass feedstock generallydepend upon the nature of the biomass. For example, for cellulosicand/or lignocellulosic feedstocks, ozone concentrations of from 0.1 g/m³to 20 g/m³ of dry feedstock provide for efficient feedstock oxidation.Typically, the water content in slurry 5050 is between 10% by weight and80% by weight (e.g., between 40% by weight and 60% by weight). Duringozone-based oxidation, the temperature of slurry 5050 can be maintainedbetween 0° C. and 100° C. to avoid violent decomposition of the ozone.

In some embodiments, feedstock slurry 5050 can be treated with anaqueous, alkaline solution that includes one or more alkali and alkalineearth hydroxides such as sodium hydroxide, potassium hydroxide, andcalcium hydroxide, and then treated thereafter with an ozone-containinggas in an oxidation reactor. This process has been observed tosignificantly increase decomposition of the biomass in slurry 5050.Typically, for example, a concentration of hydroxide ions in thealkaline solution is between 0.001% and 10% by weight of slurry 5050.After the feedstock has been wetted via contact with the alkalinesolution, the ozone-containing gas is introduced into the oxidationreactor, where it contacts and oxidizes the feedstock.

Oxidizing agents 5160 can also include other substances. In someembodiments, for example, halogen-based oxidizing agents such aschlorine and oxychlorine agents (e.g., hypochlorite) can be introducedinto slurry 5050. In certain embodiments, nitrogen-containing oxidizingsubstances can be introduced into slurry 5050. Exemplarynitrogen-containing oxidizing substances include NO and NO₂, forexample. Nitrogen-containing agents can also be combined with oxygen inslurry 5050 to create additional oxidizing agents. For example, NO andNO₂ both combine with oxygen in slurry 5050 to form nitrate compounds,which are effective oxidizing agents for biomass feedstock. Halogen- andnitrogen-based oxidizing agents can, in some embodiments, causebleaching of the biomass feedstock, depending upon the nature of thefeedstock. The bleaching can be desirable for certain biomass-derivedproducts that are extracted in subsequent processing steps.

Other oxidizing agents can include, for example, various peroxyacids,peroxyacetic acids, persulfates, percarbonates, permanganates, osmiumtetroxide, and chromium oxides.

Following oxidation preprocessing step 5060, feedstock slurry 5050 isoxidized in step 5070. If oxidizing agents 5160 were added to slurry5050 in an oxidation reactor, then oxidation proceeds in the samereactor. Alternatively, if oxidizing agents 5160 were added to slurry5050 in a preprocessing chamber, then slurry 5050 is transported to anoxidation reactor via an in-line piping system. Once inside theoxidation reactor, oxidation of the biomass feedstock proceeds under acontrolled set of environmental conditions. Typically, for example, theoxidation reactor is a cylindrical vessel that is closed to the externalenvironment and pressurized. Both batch and continuous operation ispossible, although environmental conditions are typically easier tocontrol in in-line batch processing operations.

Oxidation of feedstock slurry 5050 typically occurs at elevatedtemperatures in the oxidation reactor. For example, the temperature ofslurry 5050 in the oxidation reactor is typically maintained above 100°C., in a range from 120° C. to 240° C. For many types of biomassfeedstock, oxidation is particularly efficient if the temperature ofslurry 5050 is maintained between 150° C. and 220° C. Slurry 5050 can beheating using a variety of thermal transfer devices. For example, insome embodiments, the oxidation reactor contacts a heating bath thatincludes oil or molten salts. In certain embodiments, a series of heatexchange pipes surround and contact the oxidation reactor, andcirculation of hot fluid within the pipes heats slurry 5050 in thereactor. Other heating devices that can be used to heat slurry 5050include resistive heating elements, induction heaters, and microwavesources, for example.

The residence time of feedstock slurry 5050 in the oxidation reactor canbe varied as desired to process the feedstock. Typically, slurry 5050spends from 1 minute to 60 minutes undergoing oxidation in the reactor.For relatively soft biomass material such as lignocellulosic matter, theresidence time in the oxidation reactor can be from 5 minutes to 30minutes, for example, at an oxygen pressure of between 3 and 12 bars inthe reactor, and at a slurry temperature of between 160° C. and 210° C.For other types of feedstock, however, residence times in the oxidationreactor can be longer, e.g., as long 48 hours. To determine appropriateresidence times for slurry 5050 in the oxidation reactor, aliquots ofthe slurry can be extracted from the reactor at specific intervals andanalyzed to determine concentrations of particular products of interestsuch as complex saccharides. Information about the increase inconcentrations of certain products in slurry 5050 as a function of timecan be used to determine residence times for particular classes offeedstock material.

In some embodiments, during oxidation of feedstock slurry 5050,adjustment of the slurry pH can be performed by introducing one or morechemical agents into the oxidation reactor. For example, in certainembodiments, oxidation occurs most efficiently in a pH range of about9-11. To maintain a pH in this range, agents such as alkali and alkalineearth hydroxides, carbonates, ammonia, and alkaline buffer solutions canbe introduced into the oxidation reactor.

Circulation of slurry 5050 during oxidation can be important to ensuresufficient contact between oxidizing agents 5160 and the feedstock.Circulation of the slurry can be achieved using a variety of techniques.For example, in some embodiments, a mechanical stirring apparatus thatincludes impeller blades or a paddle wheel can be implemented in theoxidation reactor. In certain embodiments, the oxidation reactor can bea loop reactor, in which the aqueous solvent in which the feedstock issuspended is simultaneously drained from the bottom of the reactor andrecirculated into the top of the reactor via pumping, thereby ensuringthat the slurry is continually re-mixed and does not stagnate within thereactor.

After oxidation of the feedstock is complete, the slurry is transportedto a separation apparatus where a mechanical separation step 5080occurs. Typically, mechanical separation step 5080 includes one or morestages of increasingly fine filtering of the slurry to mechanicallyseparate the solid and liquid constituents.

Liquid phase 5090 is separated from solid phase 5100, and the two phasesare processed independently thereafter. Solid phase 5100 can optionallyundergo a drying step 5120 in a drying apparatus, for example. Dryingstep 5120 can include, for example, mechanically dispersing the solidmaterial onto a drying surface, and evaporating water from solid phase5100 by gentle heating of the solid material. Following drying step 5120(or, alternatively, without undergoing drying step 5120), solid phase5100 is transported for further processing steps 5140.

Liquid phase 5090 can optionally undergo a drying step 5110 to reducethe concentration of water in the liquid phase. In some embodiments, forexample, drying step 5110 can include evaporation and/or distillationand/or extraction of water from liquid phase 5090 by gentle heating ofthe liquid. Alternatively, or in addition, one or more chemical dryingagents can be used to remove water from liquid phase 5090. Followingdrying step 5110 (or alternatively, without undergoing drying step5110), liquid phase 5090 is transported for further processing steps5130, which can include a variety of chemical and biological treatmentsteps such as chemical and/or enzymatic hydrolysis.

Drying step 5110 creates waste stream 5220, an aqueous solution that caninclude dissolved chemical agents such as acids and bases in relativelylow concentrations. Treatment of waste stream 5220 can include, forexample, pH neutralization with one or more mineral acids or bases.Depending upon the concentration of dissolved salts in waste stream5220, the solution can be partially de-ionized (e.g., by passing thewaste stream through an ion exchange system). Then, the wastestream—which includes primarily water—can be re-circulated into theoverall process (e.g., as water 5150), diverted to another process, ordischarged.

Typically, for lignocellulosic biomass feedstocks following separationstep 5070, liquid phase 5090 includes a variety of soluble poly- andoligosaccharides, which can then be separated and/or reduced tosmaller-chain saccharides via further processing steps. Solid phase 5100typically includes primarily cellulose, for example, with smalleramounts of hemicellulose- and lignin-derived products.

In some embodiments, oxidation can be carried out at elevatedtemperature in a reactor such as a pyrolysis chamber. For example,referring again to FIG. 17, feedstock materials can be oxidized infilament pyrolyzer 1712. In a typical usage, an oxidizing carrier gas,e.g., air or an air/argon blend, traverses through the sample holder1713 while the resistive heating element is rotated and heated to adesired temperature, e.g., 325° C. After an appropriate time, e.g., 5 to10 minutes, the oxidized material is emptied from the sample holder. Thesystem shown in FIG. 2 can be scaled and made continuous. For example,rather than a wire as the heating member, the heating member can be anauger screw. Material can continuously fall into the sample holder,striking a heated screw that pyrolizes the material. At the same time,the screw can push the oxidized material out of the sample holder toallow for the entry of fresh, unoxidized material.

Referring again to FIG. 18, feedstock materials can be oxidized in aCurie-Point pyrolyzer 1820. In a typical usage, an oxidizing carrier gastraverses through the sample chamber 1821 while the foil 1822 isinductively heated by an applied RF field to oxidize the material at adesired temperature.

Referring again to FIG. 19, feedstock materials can be oxidized in afurnace pyrolyzer 130. In a typical usage, the sample is lowered (asindicated by arrow 137) into a hot zone 135 of furnace 132, while anoxidizing carrier gas fills the housing 136 and traverses through thesample holder 131. The sample is heated to the desired temperature for adesired time to provide an oxidized product. The oxidized product isremoved from the pyrolyzer by raising the sample holder (as indicated byarrow 134).

Referring again to FIG. 20, feedstock materials can be oxidized byforming a cellulosic target 140, along with an oxidant, such as aperoxide, and treating the target, which is housed in a vacuum chamber141, with laser light, e.g., light having a wavelength of from about 225nm to about 1600 nm. The optical configuration shown allows themonochromatic light 143 generated by the laser 142 to be directed usingmirrors 144 and 145 onto the target after passing though a lens 146 inthe vacuum chamber 141. Typically, the pressure in the vacuum chamber ismaintained at less than about 10⁻⁶ mm Hg. In some embodiments, infraredradiation is used, e.g., 1.06 micron radiation from a Nd-YAG laser. Insuch embodiments, a infrared sensitive dye can be combined with thecellulosic material to produce a cellulosic target. The infrared dye canenhance the heating of the cellulosic material. Laser treatment ofpolymers is described by Blanchet-Fincher et al. in U.S. Pat. No.5,942,649.

Referring again to FIG. 21, feedstock materials can be rapidly oxidizedby coating a tungsten filament 150, together with an oxidant, such as aperoxide, with the desired cellulosic material while the material ishoused in a vacuum chamber 151. To affect oxidation, current is passedthrough the filament, which causes a rapid heating of the filament for adesired time. Typically, the heating is continued for seconds beforeallowing the filament to cool. In some embodiments, the heating isperformed a number of times to effect the desired amount of oxidation.

Referring again to FIG. 12, in some embodiments, feedstock materials canbe oxidized with the aid of sound and/or cavitation. Generally, toeffect oxidation, the materials are sonicated in an oxidizingenvironment, such as water saturated with oxygen or another chemicaloxidant, such as hydrogen peroxide.

Referring again to FIGS. 9 and 10, in certain embodiments, ionizingradiation is used to aid in the oxidation of feedstock materials.Generally, to effect oxidation, the materials are irradiated in anoxidizing environment, such as air or oxygen. For example, gammaradiation and/or electron beam radiation can be employed to irradiatethe materials.

Other Treatment Processes

Steam explosion can be used alone without any of the processes describedherein, or in combination with any of the processes described herein.

FIG. 23 shows an overview of the entire process of converting a fibersource 400 into a product 450, such as ethanol, by a process thatincludes shearing and steam explosion to produce a fibrous material 401,which is then hydrolyzed and converted, e.g., fermented, to produce theproduct. The fiber source can be transformed into the fibrous material401 through a number of possible methods, including at least oneshearing process and at least one steam explosion process.

For example, one option includes shearing the fiber source, followed byoptional screening step(s) and optional additional shearing step(s) toproduce a sheared fiber source 402, which can then be steam exploded toproduce the fibrous material 401. The steam explosion process isoptionally followed by a fiber recovery process to remove liquids or the“liquor” 404, resulting from the steam exploding process. The materialresulting from steam exploding the sheared fiber source can be furthersheared by optional additional shearing step(s) and/or optionalscreening step(s).

In another method, the fibrous material 401 is first steam exploded toproduce a steam exploded fiber source 410. The resulting steam explodedfiber source is then subjected to an optional fiber recovery process toremove liquids, or the liquor. The resulting steam exploded fiber sourcecan then be sheared to produce the fibrous material. The steam explodedfiber source can also be subject to one or more optional screening stepsand/or one or more optional additional shearing steps. The process ofshearing and steam exploding the fiber source to produce the sheared andsteam exploded fibrous material will be further discussed below.

The fiber source can be cut into pieces or strips of confetti materialprior to shearing or steam explosion. The shearing processes can takeplace in a dry (e.g., having less than 0.25 percent by weight absorbedwater), hydrated, or even while the material is partially or fullysubmerged in a liquid, such as water or isopropanol. The process canalso optimally include steps of drying the output after steam explodingor shearing to allow for additional steps of dry shearing or steamexploding. The steps of shearing, screening, and steam explosion cantake place with or without the presence of various chemical solutions.

In a steam explosion process, the fiber source or the sheared fibersource is contacted with steam under high pressure, and the steamdiffuses into the structures of the fiber source (e.g., thelignocellulosic structures). The steam then condenses under highpressure thereby “wetting” the fiber source. The moisture in the fibersource can hydrolyze any acetyl groups in the fiber source (e.g., theacetyl groups in the hemicellulose fractions), forming organic acidssuch as acetic and uronic acids. The acids, in turn, can catalyze thedepolymerization of hemicellulose, releasing xylan and limited amountsof glucan. The “wet” fiber source (or sheared fiber source, etc.) isthen “exploded” when the pressure is released. The condensed moistureinstantaneously evaporates due to the sudden decrease in pressure andthe expansion of the water vapor exerts a shear force upon the fibersource (or sheared fiber source, etc.). A sufficient shear force willcause the mechanical breakdown of the internal structures (e.g., thelignocellulosic structures) of the fiber source.

The sheared and steam exploded fibrous material is then converted into auseful product, such as ethanol. One method of converting the fibrousmaterial is by hydrolysis to produce fermentable sugars, 412, which arethen fermented to produce the product. Other known and unknown methodsof converting fibrous materials can also be used.

In some embodiments, prior to combining the microorganism, the shearedand steam exploded fibrous material 401 is sterilized to kill anycompeting microorganisms that can be on the fibrous material. Forexample, the fibrous material can be sterilized by exposing the fibrousmaterial to radiation, such as infrared radiation, ultravioletradiation, or an ionizing radiation, such as gamma radiation. Themicroorganisms can also be killed using chemical sterilants, such asbleach (e.g., sodium hypochlorite), chlorhexidine, or ethylene oxide.One method to hydrolyze the sheared and steam exploded fibrous materialis by the use of cellulases. Cellulases are a group of enzymes that actsynergistically to hydrolyze cellulose. Commercially availableAccellerase® 1000 enzyme complex, which contains a complex of enzymesthat reduces lignocellulosic biomass into fermentable sugars can also beused.

According to current understanding, the components of cellulase includeendoglucanases, exoglucanases (cellobiohydrolases), and b-glucosidases(cellobiases). Synergism between the cellulase components exists whenhydrolysis by a combination of two or more components exceeds the sum ofthe activities expressed by the individual components. The generallyaccepted mechanism of a cellulase system (particularly of T.longibrachiatum) on crystalline cellulose is: endoglucanase hydrolyzesinternal β-1,4-glycosidic bonds of the amorphous regions, therebyincreasing the number of exposed non-reducing ends. Exoglucanases thencleave off cellobiose units from the nonreducing ends, which in turn arehydrolyzed to individual glucose units by b-glucosidases. There areseveral configurations of both endo- and exo-glucanases differing instereospecificities. In general, the synergistic action of thecomponents in various configurations is required for optimum cellulosehydrolysis. Cellulases, however, are more inclined to hydrolyze theamorphous regions of cellulose. A linear relationship betweencrystallinity and hydrolysis rates exists whereby higher crystallinityindices correspond to slower enzyme hydrolysis rates. Amorphous regionsof cellulose hydrolyze at twice the rate of crystalline regions. Thehydrolysis of the sheared and steam exploded fibrous material can beperformed by any hydrolyzing biomass process.

Steam explosion of biomass sometimes causes the formation ofby-products, e.g., toxicants, that are inhibitory to microbial andenzymatic activities. The process of converting the sheared and steamexploded fibrous material into a product can therefore optionallyinclude an overliming step prior to fermentation to precipitate some ofthe toxicants. For example, the pH of the sheared and steam explodedfibrous material can be raised to exceed the pH of 10 by adding calciumhydroxide (Ca(OH)₂) followed by a step of lowering the pH to about 5 byadding H₂SO₄. The overlimed fibrous material can then be used as iswithout the removal of precipitates. As shown in FIG. 23, the optionaloverliming step occurs just prior to the step of hydrolysis of thesheared and steam exploded fibrous material, but it is also contemplatedto perform the overliming step after the hydrolysis step and prior tothe fermenting step.

FIG. 24 depicts an example of a steam explosion apparatus 460. The steamexplosion apparatus 460 includes a reaction chamber 462, in which thefiber source and/or the fibrous material placed through a fiber sourceinlet 464. The reaction chamber is sealed by closing fiber source inletvalve 465. The reaction chamber further includes a pressurized steaminlet 466 that includes a steam valve 467. The reaction chamber furtherincludes an explosive depressurization outlet 468 that includes anoutlet valve 469 in communication with the cyclone 470 through theconnecting pipe 472. Once the reaction chamber includes the fiber sourceand/or sheared fiber source and is sealed by closing valves 465, 467 and469, steam is delivered into the reaction chamber 462 by opening thesteam inlet valve 467 allowing steam to travel through steam inlet 466.Once the reaction chamber reaches target temperature, which can takeabout 20-60 seconds, the holding time begins. The reaction temperatureis held at the target temperature for the desired holding time, whichtypically lasts from about 10 seconds to 5 minutes. At the end of theholding time period, outlet valve is open to allow for explosivedepressurization to occur. The process of explosive depressurizationpropels the contents of the reaction chamber 462 out of the explosivedepressurization outlet 468, through the connecting pipe 472, and intothe cyclone 470. The steam exploded fiber source or fibrous materialthen exits the cyclone in a sludge form into the collection bin 474 asmuch of the remaining steam exits the cyclone into the atmospherethrough vent 476. The steam explosion apparatus further includes washoutlet 478 with wash outlet valve 479 in communication with connectingpipe 472. The wash outlet valve 479 is closed during the use of thesteam explosion apparatus 460 for steam explosion, but opened during thewashing of the reaction chamber 462. The target temperature of thereaction chamber 462 is preferably between 180 and 240 degrees Celsiusor between 200 and 220 degrees Celsius. The holding time is preferablybetween 10 seconds and 30 minutes, or between 30 seconds and 10 minutes,or between 1 minute and 5 minutes.

Because the steam explosion process results in a sludge of steamexploded fibrous material, the steam exploded fibrous material canoptionally include a fiber recovery process where the “liquor” isseparated from the steam exploded fibrous material. This fiber recoverystep is helpful in that it enables further shearing and/or screeningprocesses and can allow for the conversion of the fibrous material intoa product. The fiber recovery process occurs through the use of a meshcloth to separate the fibers from the liquor. Further drying processescan also be included to prepare the fibrous material or steam explodedfiber source for subsequent processing.

Any processing technique described herein can be used at pressure aboveor below normal, earth-bound atmospheric pressure. For example, anyprocess that utilizes radiation, sonication, oxidation, pyrolysis, steamexplosion, or combinations of any of these processes to providematerials that include a carbohydrate can be performed under highpressure, which, can increase reaction rates. For example, any processor combination of processes can be performed at a pressure greater thanabout greater than 25 MPa, e.g., greater than 50 MPa, 75 MPa, 100 MPa,150 MPa, 200 MPa, 250 MPa, 350 MPa, 500 MPa, 750 MPa, 1,000 MPa, orgreater than 1,500 MPa.

Combinations of Irradiating, Sonicating, and Oxidizing Devices

In some embodiments, it can be advantageous to combine two or moreseparate irradiation, sonication, pyrolization, and/or oxidation devicesinto a single hybrid machine. For such a hybrid machine, multipleprocesses can be performed in close juxtaposition or evensimultaneously, with the benefit of increasing pretreatment throughputand potential cost savings.

For example, consider the electron beam irradiation and sonicationprocesses. Each separate process is effective in lowering the meanmolecular weight of cellulosic material by an order of magnitude ormore, and by several orders of magnitude when performed serially.

Both irradiation and sonication processes can be applied using a hybridelectron beam/sonication device as is illustrated in FIG. 25. Hybridelectron beam/sonication device 2500 is pictured above a shallow pool(depth ˜3-5 cm) of a slurry of cellulosic material 2550 dispersed in anaqueous, oxidant medium, such as hydrogen peroxide or carbamideperoxide. Hybrid device 2500 has an energy source 2510, which powersboth electron beam emitter 2540 and sonication horns 2530.

Electron beam emitter 2540 generates electron beams, which pass thoughan electron beam aiming device 2545 to impact the slurry 2550 containingcellulosic material. The electron beam aiming device can be a scannerthat sweeps a beam over a range of up to about 6 feet in a directionapproximately parallel to the surface of the slurry 2550.

On either side of the electron beam emitter 2540 are sonication horns2530, which deliver ultrasonic wave energy to the slurry 2550. Thesonication horns 2530 end in a detachable endpiece 2535 that is incontact with the slurry 2550.

The sonication horns 2530 are at risk of damage from long-term residualexposure to the electron beam radiation. Thus, the horns can beprotected with a standard shield 2520, e.g., made of lead or aheavy-metal-containing alloy such as Lipowitz metal, which is imperviousto electron beam radiation. Precautions must be taken, however, toensure that the ultrasonic energy is not affected by the presence of theshield. The detachable endpieces 2535, are constructed of the samematerial and attached to the horns 2530, are used to be in contact withthe cellulosic material 2550 and are expected to be damaged.Accordingly, the detachable endpieces 2535 are constructed to be easilyreplaceable.

A further benefit of such a simultaneous electron beam and ultrasoundprocess is that the two processes have complementary results. Withelectron beam irradiation alone, an insufficient dose can result incross-linking of some of the polymers in the cellulosic material, whichlowers the efficiency of the overall depolymerization process. Lowerdoses of electron beam irradiation and/or ultrasound radiation can alsobe used to achieve a similar degree of depolymerization as that achievedusing electron beam irradiation and sonication separately.

An electron beam device can also be combined with one or more ofhigh-frequency, rotor-stator devices, which can be used as analternative to ultrasonic energy devices, and performs a similarfunction.

Further combinations of devices are also possible. For example, anionizing radiation device that produces gamma radiation emitted from,e.g., ⁶⁰Co pellets, can be combined with an electron beam source and/oran ultrasonic wave source.

The radiation devices for pretreating biomass discussed above can alsobe combined with one or more devices that perform one or more pyrolysisprocessing sequences. Such a combination can again have the advantage ofhigher throughput. Nevertheless, caution must be observed, as there canbe conflicting requirements between some radiation processes andpyrolysis. For example, ultrasonic radiation devices can require thefeedstock be immersed in a liquid oxidizing medium. On the other hand,as discussed previously, it can be advantageous for a sample offeedstock undergoing pyrolysis to be of a particular moisture content.In this case, the new systems automatically measure and monitor for aparticular moisture content and regulate the same Further, some or allof the above devices, especially the pyrolysis device, can be combinedwith an oxidation device as discussed previously.

Primary Processes (Processing Treated Biomass) Fermentation

Generally, various microorganisms can produce a number of usefulproducts by operating on, e.g., fermenting treated biomass materials.For example, alcohols, organic acids, hydrocarbons, hydrogen, proteinsor mixtures of any of these materials can be produced by fermentation orother processes.

The microorganism can be a natural microorganism or an engineeredmicroorganism. For example, the microorganism can be a bacterium, e.g.,a cellulolytic bacterium, a fungus, e.g., a yeast, a plant or a protist,e.g., an algae, a protozoa or a fungus-like protist, e.g., a slime mold.When the organisms are compatible, mixtures of organisms can beutilized.

To aid in the breakdown of the treated biomass materials that includecellulose, one or more enzymes, e.g., a cellulolytic enzyme can beutilized. In some embodiments, materials that include cellulose arefirst treated with the enzyme, e.g., by combining the materials and theenzyme in an aqueous solution. This material can then be combined withthe microorganism. In other embodiments, the materials that include thecellulose, the one or more enzymes and the microorganism are combinedconcurrently, e.g., by combining in an aqueous solution.

Also, to aid in the breakdown of the treated biomass materials, thetreated biomass materials can be further treated (e.g., postirradiation) with heat, a chemical (e.g., mineral acid, base or a strongoxidizer such as sodium hypochlorite), and/or an enzyme.

During fermentation, sugars released from cellulolytic hydrolysis orsaccharification, are fermented to, e.g., ethanol, by a fermentingmicroorganism such as yeast. Suitable fermenting microorganisms have theability to convert carbohydrates, such as glucose, xylose, arabinose,mannose, galactose, oligosaccharides, or polysaccharides intofermentation products. Fermenting microorganisms include strains of thegenus Saccharomyces spp. e.g., Saccharomyces cerevisiae (baker's yeast),Saccharomyces distaticus, and Saccharomyces uvarum; the genusKluyveromyces, e.g., species Kluyveromyces marxianus, and Kluyveromycesfragilis; the genus Candida, e.g., Candida pseudotropicalis, and Candidabrassicae; the genus Clavispora, e.g., species Clavispora lusitaniae andClavispora opuntiae; the genus Pachysolen, e.g., species Pachysolentannophilus; the genus Bretannomyces, e.g., species Bretannomycesclausenii; the genus Pichia, e.g., species Pichia stipitis; and thegenus Saccharophagus, e.g., species Saccharophagus degradans(Philippidis, 1996, “Cellulose Bioconversion Technology”, in Handbook onBioethanol: Production and Utilization, Wyman, ed., Taylor & Francis,Washington, D.C., 179-212).

Commercially available yeast include, for example, Red Star®/LesaffreEthanol Red (available from Red Star/Lesaffre, USA); FALI® (availablefrom Fleischmann's Yeast, a division of Burns Philip Food Inc., USA);SUPERSTART® (available from Alltech, now Lallemand); GERT STRAND®(available from Gert Strand AB, Sweden); and FERMOL® (available from DSMSpecialties).

Bacteria that can ferment biomass to ethanol and other products include,e.g., Zymomonas mobilis and Clostridium thermocellum (Philippidis, 1996,supra). Leschine et al. (International Journal of Systematic andEvolutionary Microbiology 2002, 52, 1155-1160) describe an anaerobic,mesophilic, cellulolytic bacterium from forest soil, Clostridiumphytofermentans sp. nov., which converts cellulose to ethanol.

Fermentation of biomass to ethanol and other products can be carried outusing certain types of thermophilic or genetically engineeredmicroorganisms, such as Thermoanaerobacter species, including T.mathranii, and yeast species such as Pichia species. An example of astrain of T. mathranii is A3M4 described in Sonne-Hansen et al. (AppliedMicrobiology and Biotechnology 1993, 38, 537-541) or Ahring et al.(Arch. Microbiol. 1997, 168, 114-119).

Yeast and Zymomonas bacteria can be used for fermentation or conversion.The optimum pH for yeast is from about pH 4 to 5, while the optimum pHfor Zymomonas is from about pH 5 to 6. Typical fermentation times areabout 24 to 96 hours with temperatures in the range of 26° C. to 40° C.,however thermophilic microorganisms prefer higher temperatures.

Enzymes that break down biomass, such as cellulose, to lower molecularweight carbohydrate-containing materials, such as glucose, are referredto as cellulolytic enzymes or cellulase; this process is referred to an“saccharification”. These enzymes can be a complex of enzymes that actsynergistically to degrade crystalline cellulose. Examples ofcellulolytic enzymes include: endoglucanases, cellobiohydrolases, andcellobiases (β-glucosidases). For example, cellulosic substrate isinitially hydrolyzed by endoglucanases at random locations producingoligomeric intermediates. These intermediates are then substrates forexo-splitting glucanases such as cellobiohydrolase to produce cellobiosefrom the ends of the cellulose polymer. Cellobiose is a water-solubleβ-1,4-linked dimer of glucose. Finally cellobiase cleaves cellobiose toyield glucose.

A cellulase is capable of degrading biomass and can be of fungal orbacterial origin. Suitable enzymes include cellulases from the generaBacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium,Chrysosporium and Trichoderma, and include species of Humicola,Coprinus, Thielavia, Fusarium, Myceliophthora, Acremonium,Cephalosporium, Scytalidium, Penicillium or Aspergillus (see, e.g., hsEP458162), especially those produced by a strain selected from the speciesHumicola insolens (reclassified as Scytalidium thermophilum, see, e.g.,U.S. Pat. No. 4,435,307), Coprinus cinereus, Fusarium oxysporum,Myceliophthora thermophila, Meripilus giganteus, Thielavia terrestris,Acremonium sp., Acremonium persicinum, Acremonium acremonium, Acremoniumbrachypenium, Acremonium dichromosporum, Acremonium obclavatum,Acremonium pinkertoniae, Acremonium roseogriseum, Acremoniumincoloratum, and Acremonium furatum; preferably from the speciesHumicola insolens DSM 1800, Fusarium oxysporum DSM 2672, Myceliophthorathermophila CBS 117.65, Cephalosporium sp. RYM-202, Acremonium sp. CBS478.94, Acremonium sp. CBS 265.95, Acremonium persicinum CBS 169.65,Acremonium acremonium AHU 9519, Cephalosporium sp. CBS 535.71,Acremonium brachypenium CBS 866.73, Acremonium dichromosporum CBS683.73, Acremonium obclavatum CBS 311.74, Acremonium pinkertoniae CBS157.70, Acremonium roseogriseum CBS 134.56, Acremonium incoloratum CBS146.62, and Acremonium furatum CBS 299.70H. Cellulolytic enzymes canalso be obtained from Chrysosporium, preferably a strain ofChrysosporium lucknowense. Additionally, Trichoderma (particularlyTrichoderma viride, Trichoderma reesei, and Trichoderma koningii),alkalophilic Bacillus (see, for example, U.S. Pat. No. 3,844,890 and EP458162), and Streptomyces (see, e.g., EP 458162) can be used.

Cellulolytic enzymes produced using recombinant technology can also beused (see, e.g., WO 2007/071818 and WO 2006/110891).

The cellulolytic enzymes used can be produced by fermentation of theabove-noted microbial strains on a nutrient medium containing suitablecarbon and nitrogen sources and inorganic salts, using procedures knownin the art (see, e.g., Bennettand LaSure (eds.), More Gene Manipulationsin Fungi, Academic Press, CA 1991). Suitable media are available fromcommercial suppliers or can be prepared according to publishedcompositions (e.g., in catalogues of the American Type CultureCollection). Temperature ranges and other conditions suitable for growthand cellulase production are known in the art (see, e.g., Bailey andOllis, Biochemical Engineering Fundamentals, McGraw-Hill Book Company,NY, 1986).

Treatment of cellulose with cellulase is usually carried out attemperatures between 30° C. and 65° C. Cellulases are active over arange of pH of about 3 to 7. A saccharification step can last e.g., upto 120 hours. The cellulase enzyme dosage achieves a sufficiently highlevel of cellulose conversion. For example, an appropriate cellulasedosage is typically between 5.0 and 50 Filter Paper Units (FPU or IU)per gram of cellulose. The FPU is a standard measurement and is definedand measured according to Ghose (1987, Pure and Appl. Chem. 59:257-268).

In particular embodiments, ACCELERASE™ 1000 (GENENCOR) is utilized asthe enzyme system at a loading of 0.25 mL per gram of substrate.ACCELLERASE® 1000 enzyme complex is a multiple enzyme cocktail withmultiple activities, mainly exoglucanase, endoglucanase, hemicellulaseand beta-glucosidase. The cocktail has a minimum endoglucanase activityof 2500 CMC U/g and a minimum beta-glucosidase activity of 400 pNPG U/g.The pH of the cocktail is from about 4.8 to about 5.2. In otherparticular embodiments, the enzyme system utilized is a blend ofCELLUCLAST® 1.5 L and Novozyme 188. For example, 0.5 mL of CELLUCLAST®1.5 L and 0.1 mL of Novozyme 188 can be used for each gram of substrate.When a higher hemicellulase (xylanase) activity is desired, OPTIMASH™ BGcan be utilized.

Gasification

In addition to using pyrolysis for pre-treatment of feedstock, pyrolysiscan also be used to process pre-treated feedstock to extract usefulmaterials. In particular, a form of pyrolysis known as gasification canbe employed to generate fuel gases along with various other gaseous,liquid, and solid products. To perform gasification, the pre-treatedfeedstock is introduced into a pyrolysis chamber and heated to a hightemperature, typically 700° C. or more. The temperature used dependsupon a number of factors, including the nature of the feedstock and thedesired products.

Quantities of oxygen (e.g., as pure oxygen gas and/or as air) and steam(e.g., superheated steam) are also added to the pyrolysis chamber tofacilitate gasification. These compounds react with carbon-containingfeedstock material in a multiple-step reaction to generate a gas mixturecalled synthesis gas (or “syngas”). Essentially, during gasification, alimited amount of oxygen is introduced into the pyrolysis chamber toallow some feedstock material to combust to form carbon monoxide andgenerate process heat. The process heat can then be used to promote asecond reaction that converts additional feedstock material to hydrogenand carbon monoxide.

In a first step of the overall reaction, heating the feedstock materialproduces a char that can include a wide variety of differenthydrocarbon-based species. Certain volatile materials can be produced(e.g., certain gaseous hydrocarbon materials), resulting in a reductionof the overall weight of the feedstock material. Then, in a second stepof the reaction, some of the volatile material that is produced in thefirst step reacts with oxygen in a combustion reaction to produce bothcarbon monoxide and carbon dioxide. The combustion reaction releasesheat, which promotes the third step of the reaction. In the third step,carbon dioxide and steam (e.g., water) react with the char generated inthe first step to form carbon monoxide and hydrogen gas. Carbon monoxidecan also react with steam, in a water gas shift reaction, to form carbondioxide and further hydrogen gas.

Gasification can be used as a primary process to generate productsdirectly from pre-treated feedstock for subsequent transport and/orsale, for example. Alternatively, or in addition, gasification can beused as an auxiliary process for generating fuel for an overallprocessing system. The hydrogen-rich syngas that is generated via thegasification process can be burned, for example, to generate electricityand/or process heat that can be directed for use at other locations inthe processing system. As a result, the overall processing system can beat least partially self-sustaining A number of other products, includingpyrolysis oils and gaseous hydrocarbon-based substances, can also beobtained during and/or following gasification; these can be separatedand stored or transported as desired.

A variety of different pyrolysis chambers are suitable for gasificationof pre-treated feedstock, including the pyrolysis chambers disclosedherein. In particular, fluidized bed reactor systems, in which thepre-treated feedstock is fluidized in steam and oxygen/air, providerelatively high throughput and straightforward recovery of products.Solid char that remains following gasification in a fluidized bed system(or in other pyrolysis chambers) can be burned to generate additionalprocess heat to promote subsequent gasification reactions.

Processing Treated Biomass Distillation

After fermentation, the resulting fluids can be distilled using, forexample, a “beer column” to separate ethanol and other alcohols from themajority of water and residual solids. The vapor exiting the beer columncan be 35% by weight ethanol and fed to a rectification column. Amixture of nearly azeotropic (92.5%) ethanol and water from therectification column can be purified to pure (99.5%) ethanol usingvapor-phase molecular sieves. The beer column bottoms can be sent to thefirst effect of a three-effect evaporator. The rectification columnreflux condenser can provide heat for this first effect. After the firsteffect, solids can be separated using a centrifuge and dried in a rotarydryer. A portion (25%) of the centrifuge effluent can be recycled tofermentation and the rest sent to the second and third evaporatoreffects. Most of the evaporator condensate can be returned to theprocess as fairly clean condensate with a small portion split off towaste water treatment to prevent build-up of low-boiling pointcompounds.

Waste Water Treatment

Wastewater treatment is used to minimize makeup water requirements ofthe plant by treating process water for reuse within the plant.Wastewater treatment can also produce fuel (e.g., sludge and biogas)that can be used to improve the overall efficiency of the ethanolproduction process. For example, as described in further detail below,sludge and biogas can be used to create steam and electricity that canbe used in various plant processes.

Wastewater is initially pumped through a screen (e.g., a bar screen) toremove large particles, which are collected in a hopper. In someembodiments, the large particles are sent to a landfill. Additionally oralternatively, the large particles are burned to create steam and/orelectricity as described in further detail below. In general, thespacing on the bar screen is between ¼ inch to 1 inch spacing (e.g., ½inch spacing).

The wastewater then flows to an equalization tank, where the organicconcentration of the wastewater is equalized during a retention time. Ingeneral, the retention time is between 8 hours and 36 hours (e.g., 24hours). A mixer is disposed within the tank to stir the contents of thetank. In some embodiments, a plurality of mixers disposed throughout thetank are used to stir the contents of the tank. In certain embodiments,the mixer substantially mixes the contents of the equalization tank suchthat conditions (e.g., wastewater concentration and temperature)throughout the tank are uniform.

A first pump moves water from the equalization tank through aliquid-to-liquid heat exchanger. The heat exchanger is controlled (e.g.,by controlling the flow rate of fluid through the heat exchanger) suchthat wastewater exiting the heat exchanger is at a desired temperaturefor anaerobic treatment. For example, the desired temperature foranaerobic treatment can be between 40° C. to 60° C.

After exiting the heat exchanger, the wastewater enters one or moreanaerobic reactors. In some embodiments, the concentration of sludge ineach anaerobic reactor is the same as the overall concentration ofsludge in the wastewater. In other embodiments, the anaerobic reactorhas a higher concentration of sludge than the overall concentration ofsludge in the wastewater.

A nutrient solution containing nitrogen and phosphorus is metered intoeach anaerobic reactor containing wastewater. The nutrient solutionreacts with the sludge in the anaerobic reactor to produce biogas whichcan contain 50% methane and have a heating value of approximately 12,000British thermal units, or Btu, per pound). The biogas exits eachanaerobic reactor through a vent and flows into a manifold, where aplurality of biogas streams are combined into a single stream. Acompressor moves the stream of biogas to a boiler or a combustion engineas described in further detail below. In some embodiments, thecompressor also moves the single stream of biogas through adesulphurization catalyst. Additionally or alternatively, the compressorcan move the single stream of biogas through a sediment trap.

A second pump moves anaerobic effluent from the anaerobic reactors toone or more aerobic reactors (e.g., activated sludge reactors). Anaerator is disposed within each aerobic reactor to mix the anaerobiceffluent, sludge, oxygen (e.g., oxygen contained in air). Within eachaerobic reactor, oxidation of cellular material in the anaerobiceffluent produces carbon dioxide, water, and ammonia.

Aerobic effluent moves (e.g., via gravity) to a separator, where sludgeis separated from treated water. Some of the sludge is returned to theone or more aerobic reactors to create an elevated sludge concentrationin the aerobic reactors, thereby facilitating the aerobic breakdown ofcellular material in the wastewater. A conveyor removes excess sludgefrom the separator. As described in further detail below, the excesssludge is used as fuel to create steam and/or electricity.

The treated water is pumped from the separator to a settling tank.Solids dispersed throughout the treated water settle to the bottom ofthe settling tank and are subsequently removed. After a settling period,treated water is pumped from the settling tank through a fine filter toremove any additional solids remaining in the water. In someembodiments, chlorine is added to the treated water to kill pathogenicbacteria. In some embodiments, one or more physical-chemical separationtechniques are used to further purify the treated water. For example,treated water can be pumped through a carbon adsorption reactor. Asanother example, treated water can pumped through a reverse osmosisreactor.

Waste Combustion

The production of alcohol from biomass can result in the production ofvarious by-product streams useful for generating steam and electricityto be used in other parts of the plant. For example, steam generatedfrom burning by-product streams can be used in the distillation process.As another example, electricity generated from burning by-productstreams can be used to power electron beam generators and ultrasonictransducers used in pretreatment.

The by-products used to generate steam and electricity are derived froma number of sources throughout the process. For example, anaerobicdigestion of wastewater produces a biogas high in methane and a smallamount of waste biomass (sludge). As another example, post-distillatesolids (e.g., unconverted lignin, cellulose, and hemicellulose remainingfrom the pretreatment and primary processes) can be used as a fuel.

The biogas is diverted to a combustion engine connected to an electricgenerator to produce electricity. For example, the biogas can be used asa fuel source for a spark-ignited natural gas engine. As anotherexample, the biogas can be used as a fuel source for a direct-injectionnatural gas engine. As another example, the biogas can be used as a fuelsource for a combustion turbine. Additionally or alternatively, thecombustion engine can be configured in a cogeneration configuration. Forexample, waste heat from the combustion engines can be used to providehot water or steam throughout the plant.

The sludge and post-distillate solids can be burned to heat waterflowing through a heat exchanger. In some embodiments, the water flowingthrough the heat exchanger is evaporated and superheated to steam. Incertain embodiments, the steam is used in the pretreatment rector and inheat exchange in the distillation and evaporation processes.Additionally or alternatively, the steam expands to power a multi-stagesteam turbine connected to an electric generator. Steam exiting thesteam turbine is condensed with cooling water and returned to the heatexchanger for reheating to steam. In some embodiments, the flow rate ofwater through the heat exchanger is controlled to obtain a targetelectricity output from the steam turbine connected to an electricgenerator. For example, water can be added to the heat exchanger toensure that the steam turbine is operating above a threshold condition(e.g., the turbine is spinning fast enough to turn the electricgenerator).

While certain embodiments have been described, other embodiments arepossible.

As an example, while the biogas is described as being diverted to acombustion engine connected to an electric generator, in certainembodiments, the biogas or some portion thereof can also be passedthrough a fuel reformer to produce hydrogen. The hydrogen is thenconverted to electricity through a fuel cell.

As another example, while the biogas is described as being burned apartfrom the sludge and post-distillate solids, in certain embodiments, someor all of the waste by-products can be burned together to produce steam.

Products/Co-Products

In some embodiments, the present invention provides materials generatedusing the methods described herein. In some cases, such materials can beused in the absence of materials added to the biomass pre or postprocessing, e.g., materials that are not naturally present in biomass.In such cases, the materials will contain naturally occurring materials,e.g., derived from biomass. Alternatively or in addition, the materialsgenerated using the methods described herein can be combined with othernatural and/or synthetic materials, e.g., materials that are notnaturally present in biomass.

As described above, in some embodiments, the methods described hereincan be used for converting (e.g., fermenting) biomass to an energyproduct (e.g., an alcohol such as ethanol or a hydrocarbon) and/or otherproducts that result from the conversion process (e.g., organic acids).In such cases, the biomass will be exposed to conditions suitable forsuch a conversion. Exemplary conditions can include, e.g., at leastbiomass and one or more microorganisms capable of converting the biomassto energy (e.g., an alcohol) in an environment suitable for thoseorganisms to function. This conversion process can be allowed to proceedto a point where at least a portion of the biomass is converted toenergy (e.g., ethanol) and/or other products that result from theconversion process (e.g., as described below) and/or to a point whereall (e.g., essentially all) of the materials are converted to energy(e.g., ethanol) and/or other products that result from the conversionprocess. For example, at least about 10, 20, 30, 40, 50, 60, 70, 80, 90,95, 98, 99, 99.5, or 100% of the materials exposed to conditionssuitable for fermentation is converted to energy (e.g., ethanol) and/orother products that result from the conversion process.

Alternatively or in addition, the methods described herein can be usedto modify biomass, e.g., to modify (e.g., increase, decrease, ormaintain) the solubility of the native materials, to change thestructure of, e.g., to functionalize, the native materials, and/or alter(e.g., lower) the molecular weight and/or crystallinity relative to anative material. Such methods can be performed together or alone. Forexample, the methods described herein can be used to convert a portionof the biomass to energy. The methods described herein can also be usedto modify (e.g., increase, decrease, or maintain) the solubility, tochange the structure, e.g., functionalize, and/or alter (e.g., lower)the molecular weight and/or crystallinity of the biomass, or vice versa.

In some embodiments, the methods described herein can be used to obtain(e.g., extract, isolate, and/or increase the availability of, e.g., ascompared to unprocessed biomass materials) one or more componentscontained in an unprocessed biomass material (e.g., a raw material).Exemplary components that can be obtained (e.g., extracted, isolated,and/or increased in availability (e.g., compared to unprocessed biomassmaterials)) include, but are not limited to sugars (e.g., 1,4-diacids(e.g., succinic acids, fumaric acids, and malic acids), 2,5-furandicarboxylic acids, 3-hydroxy propionic acid, aspartic acid, glucaricacid, glutamic acid, itaconic acid, 3-hydroxy propionic acid, asparticacid, glucaric acid, glutamic acid, itaconic acid, levulinic acid,3-hydroxybutyrolactone, glycerol, sorbitol, and/or xylitol/aribitol),dextrins, cyclodextrins, amylase, amylopectin, germ, proteins, aminoacids, peptides, nucleic acids, fats, lipids, fatty acids, gluten,sweeteners (e.g., glucose), sugar alcohols (e.g., arabitol, xylitol,ribitol, mannitol, sorbitol, isomalt, maltitol, and lactitol), oils(e.g., triglyceride vegetable oils (e.g., soybean oil, palm oil,rapeseed oil, sunflower seed oil, peanut oil, cottonseed oil, palmkernel oil, olive oil), corn oil, oat oil, nut oil, and palm oil),minerals, vitamins, toxins, and other chemicals, ash, and flavenoids.Such components can be used in various application described below,e.g., as individual components, in combination with one or moreadditional components, in combination with processed and/or unprocessedbiomass, and/or in combination with one or more additional componentsnot obtained (e.g., extracted, isolated, and/or increased inavailability) from biomass. Methods for obtaining one or more of thesecomponents are known in the art.

In some embodiments, the methods described herein can be used toincrease the availability of one or more components contained in biomass(e.g., unprocessed and/or partially processed biomass). Components withincreased availability can be more readily obtained (e.g., extractedand/or isolated), more readily used, and/or can be more readily amenableto an animal (e.g., digested or absorbed by an animal). Components withincreased availability can include, for example, components that occurnaturally in biomass and/or components that are generated using themethods described herein (e.g., cross-linked species, low molecularweight species). Such components can increase the value of biomass. Forexample, low molecular weight species are more readily hydrolyzed in thestomach than unprocessed biomass. Thus, biomass containing more readilyavailable low molecular weight species can be used as a more valuablefood source, e.g., for animals or insects, or for use in agriculture,aquaculture, e.g., the cultivation of fish, aquatic microorganisms,aquatic plants, seaweed and algae.

In some embodiments, the methods described herein can be used tosterilize biomass to render the materials suitable for consumption byanimals and/or humans (e.g., ingestion or implantation), by insects, orfor use in agriculture, aquaculture, e.g., the cultivation of fish,aquatic microorganisms, aquatic plants, seaweed and algae. In someembodiments, irradiation treatment of cellulosic material will renderthe biomass sterile and, therefore, suitable for consumption in animalsand/or humans (e.g., ingestion or implantation). The irradiatedcellulose can also be used in other products or co-products.

In some embodiments, the methods described herein can be used to processbiomass into a material intended for consumption (e.g., ingestion orimplantation) in humans and/or non-human animals. Generally, suchmaterials should be essentially free of infectious material (e.g.,pathogenic and/or non-pathogenic material), toxins, and/or othermaterials (e.g., bacterial and fungal spores, insects, and larvae) thatcan be harmful to the human and/or animal. Methods known in the artand/or described herein can be used to remove, inactivate, and/orneutralize infectious material (e.g., pathogenic and/or non-pathogenicmaterial) and/or toxins that can be harmful to humans and/or animals orthat are generally undesirable in a material intended for use in humansand/or animals. For example, the methods can be used to remove,inactivate, and/or neutralize infectious material that can be present inthe biomass. Such materials include, e.g., pathogenic and non-pathogenicbacteria, viruses, fungus, parasites, and prions (e.g., infectiousproteins). In some instances, the methods described herein can be usedto remove, inactivate, and/or neutralize toxins, e.g., bacterial toxinsand plant toxins. Alternatively or in addition, the methods describedherein can be used to remove, inactivate, and/or neutralize materialsthat can be present in the biomass that are not necessarily harmful, butare undesirable in a material to be used in humans and/or animals or inagriculture or aquaculture. Exemplary materials include, but are notlimited to, bacterial and fungal spores, insects, and larvae.

In some embodiments, the methods described herein can be used to producethe products and co-products and bioconversion products described hereinin challenging environments. Such environments can include environmentsthat present space limitations and/or extreme environmental conditions,for example, locations with excessive heat or cold, locations withexcessive radiation, locations with excessive pollutants, and/orlocations with limited oxygen supply or sunlight. In some embodiments,such environments can include, but are not limited to, for example, onboard space craft, on board space stations (e.g., extraterrestriallocations), on board submarines (e.g., nuclear submarines) and othermarine vessels or barges or platforms designed to remain at sea forextensive time periods, submarine locations (e.g., civilian and/ormilitary underwater facilities), desert environments, polarenvironments, subzero environments (e.g., permafrozen locations),elevated environments (e.g., where oxygen supplies can be limited and/orextreme temperatures are present), and remote locations (e.g., selfcontained locations).

In some embodiments, the products and/or co-products described herein,e.g., resulting from the treatment of biomass using the methodsdescribed herein can be, e.g., solids (e.g., particulates (e.g., films),granulates, and/or powders), semi-solids, liquids, gasses, vapors, gels,and combinations thereof.

Alcohols

Alcohols produced using the materials described herein can include, butare not limited to, a monohydroxy alcohol, e.g., ethanol, or apolyhydroxy alcohol, e.g., ethylene glycol or glycerin. Examples ofalcohols that can be produced include, but are not limited to, methanol,ethanol, propanol, isopropanol, butanol, e.g., n-, sec- or t-butanol,ethylene glycol, propylene glycol, 1,4-butane diol, glycerin or mixturesof these alcohols.

In some embodiments, the alcohols produced using the treatment methodsdisclosed herein can be used in the production of a consumable beverage.

Hydrocarbons

Hydrocarbons include aromatic hydrocarbons or arenes, alkanes, alkenesand alkynes. Exemplary hydrocarbons include methane, ethane, propane,butane, isobutene, hexane, heptane, isobutene, octane, iso-octane,nonane, decane, benzene and toluene.

Organic Acids

The organic acids produced using the methods and materials describedherein can include monocarboxylic acids or polycarboxylic acids.Examples of organic acids include formic acid, acetic acid, propionicacid, butyric acid, valeric acid, caproic, palmitic acid, stearic acid,oxalic acid, malonic acid, succinic acid, glutaric acid, oleic acid,linoleic acid, glycolic acid, lactic acid, γ-hydroxybutyric acid, ormixtures of these acids.

Foodstuffs

As described herein, the present invention provides methods useful formodifying biomass, e.g., by modifying (e.g., increasing, decreasing, ormaintaining) the solubility of the native materials, changing thestructure of (e.g., functionalizing) the native materials, and/oraltering (e.g., lowering) the molecular weight and/or crystallinityrelative to a native material. The methods can be used to preparematerials with properties that can be favorable for use as or in theproduction of a foodstuff. For example, the methods can be used toprepare a material with improved (e.g., increased or decreased)solubility, e.g., compared to a native material, which can be used as amore easily absorbed foodstuff. Increased solubility can be assessed,e.g., by dispersing (e.g., dissolving) unprocessed and processedmaterials in a suitable solvent, removing undissolved material,detecting the materials and/or specific components of the materials(e.g., sugars), and comparing the levels of the detected materials inthe processed and unprocessed materials. In some cases, the solventcontaining the materials can be modified, e.g., by heating or byadjusting the pH.

Alternatively or in addition, the methods can be used to prepare amaterial with a higher nutritional value (e.g., higher energy (e.g.,more digestively available food energy) and/or nutrient availability)when the material is ingested by an animal, e.g., compared to a nativematerial or unprocessed biomass. Such methods will not necessarilyincrease the total amount of energy or nutrients present in a set amount(e.g., weight) of a specific type of processed biomass compared to thesame amount and type of unprocessed biomass. Rather, the methodsdescribed herein can be used to increase the nutritional value (e.g.,the availability of energy and/or one or more nutrients) in a set amount(e.g., weight) of a specific type of processed biomass compared to thesame amount and type of unprocessed biomass.

Increasing the availability of food energy of a particular type ofbiomass can be used to increase the metabolizable energy intake (MEI) ofthat biomass. Methods for measuring food energy are known in the art.MEI is typically calculated by multiplying the number of kilocalories orkilojoules contained in a food item by 85%. In some embodiments, themethods described herein can be used to increase the MEI of biomass.

Methods for comparing the MEI of processed and unprocessed biomass caninclude, for example, feeding equal amounts of processed or unprocessedbiomass to at least two distinct groups of one or more animals, andmeasuring the growth response of the animals.

Nutrient availability can be assessed by conducting a digestion trial.Protocols for conducting digestion trials are known in the art. Forexample, total nutrient levels can be determined in processed and/orunprocessed biomass. Equal amounts of processed or unprocessed biomasscan be fed to at least two distinct groups of one or more animals. Fecalloss of one or more nutrients is then determined for a defined period oftime. Increased nutrient availability is defined as lower amounts of oneor more nutrients in the animal feces. Alternatively or in addition,nutrient availability can be assessed by measuring and comparing thelevels of one or more nutrients in the blood of animals fed processedand unprocessed biomass.

In some embodiments, the nutritional value of biomass can be increasedby increasing the digestibility of one or more of, food energy,carbohydrates, sugars, proteins, fats (saturated, monounsaturated, andpolyunsaturated), cholesterol, dietary fiber, vitamins (e.g., vitamin A,E, C, B6, B12, carotene, thiamin, riboflavin, and niacin), minerals(e.g., calcium, phosphorus, magnesium, iron, zinc, copper, potassium,selenium, and sodium), and oils when the biomass is ingested by ananimal.

In general, the methods described herein can be selected and/oroptimized to select a method or combination of methods that result inthe most readily soluble, absorbable, and/or digestible material, e.g.,with a desired nutrient availability (e.g., a higher nutrient (e.g.,protein, amino acid, carbohydrate, mineral, vitamin, fat lipid, and oil)availability than native unprocessed material) that can be used inhumans and/or animals as a foodstuff. Because the biomass materials arereadily available and cheap, the materials resulting from such methodswill provide an economical foodstuff and reduce waste.

In some embodiments, the materials and methods described herein can beused in the production of a foodstuff, e.g., agricultural foodstuffs andfoodstuffs suitable for ingestion by mammals, birds, and/or fish. Suchanimals include, but are not limited to food production animals,domestic animals, zoo animals, laboratory animals, and/or humans.

In some embodiments, materials produced by the methods described hereinthat are intended for use as foodstuffs (e.g., in humans and/or animals)can be additionally processed, e.g., hydrolyzed. Hydrolization methodsare known in the art and include, for example, the use of enzymes,acids, and/or bases to reduce the molecular weight of saccharides. Insome embodiments, foodstuffs resulting from the methods described hereincan include enzymes (e.g., dried enzymes, active enzymes, and/or enzymesrequiring activation).

In some embodiments, materials produced by the methods described hereinthat are intended for use as foodstuffs (e.g., in humans and/or animals)can be additionally processed to increase sterility of the materialsand/or remove, inactivate, and/or neutralize materials that can bepresent in the biomass, e.g., infectious material (e.g., pathogenicand/or non-pathogenic material), toxins, and/or other materials (e.g.,bacterial and fungal spores, insects, and larvae). In general, themethods described herein can be selected and optimized in order topromote optimal removal, inactivation, and/or neutralization ofmaterials that may be undesirable.

Animal Foodstuffs

In excess of 600 million tons of animal foodstuff is produced annuallyaround the world with an annual growth rate of about 2%. Agriculture isone of the largest consumers of animal foodstuffs with farmers in theUnited States spending in excess of $20 billion dollars per year onagricultural foodstuffs for food producing animals. Other foodstuffsconsumers include, for example, pet owners, zoos, and laboratories thatkeep animals for research studies.

In general, an animal foodstuff should meet or exceed the specificrequirements of a target animal, e.g., to maintain or improve the healthof a specific type or species of animal, promote the growth of a targetanimal (e.g., tissue gain), and/or to promote food production. Improvedanimal foodstuffs (e.g., more soluble, absorbable, and/or digestiblefoodstuffs) will promote or support these same effects using a smalleramount of foodstuff and/or for a lower cost.

Currently used raw materials in commercially prepared foodstuff includefeed grains (e.g., corn, soybean, sorghum, oats, and barley). The feedindustry is the largest purchaser of U.S. corn, feed grains, and soybeanmeal. However, with the escalating price of feed grains such as corn,cheaper alternatives are desired. The most abundantly availablefoodstuff is biomass, e.g., cellulosic material. In some embodiments,the methods described herein can be used to increase the nutrientavailability of any of these materials, e.g., to maintain or improve thehealth of a specific type or species of animal, promote the growth of atarget animal (e.g., tissue gain), and/or to promote food production.The low nutrient availability of commonly used foodstuffs (e.g., hay andgrasses) is largely attributed to the high cellulose, hemicellulose, andlignin content of such material. Unlike humans, who cannot digestcellulose, herbivores, e.g., ruminants, are capable of digestingcellulose, at least partially, through a process known as rumination.This process, however, is inefficient and requires multiple rounds ofregurgitation. For example, ruminants only digest about 30-50 percent ofthe cellulose and hemicellulose. In some embodiments, the methodsdescribed herein can be used to increase the nutrient availability ornutritional value of any of these materials, e.g., to maintain orimprove the health of a specific type or species of animal, promote thegrowth of a target animal (e.g., tissue gain), and/or to promote foodproduction. The methods described herein use reduced amounts offoodstuffs, at a lower cost, and/or with less waste.

Generally, increasing the nutrient availability of an animal foodstuffwill reduce the amount of feed required to be fed to an animal for theanimal to receive the same amount of energy. Consequently, the animalwill require less foodstuff thus providing a more economical foodstuff.

Various techniques have been attempted to increase the nutrientavailability of a foodstuff with limited success. Such techniquesinclude the use of enzymes, such as cellulosic enzymes, to break downcellulosic material into shorter chain oligosaccharides, which can bemore readily digested. Although used in Europe and Australia, thispractice is expensive and not widely used in developing countries. Othertechniques include removing stover to prevent leaf loss, air removal,physically treating the material (e.g., compacting cellulosic material,reducing particle size, and fine grinding), chemical treatment, andoverfeeding. Additionally, foodstuffs composed largely from cellulosicmaterial are frequently supplemented with nutrient systems (e.g.,premixes). These nutrient systems are typically designed to provide thenutritional requirements of a target animal. Despite ensuring theanimals receive the required nutrients, such systems do not makeefficient use of the cellulosic material.

The methods described herein provide methods for improving the nutrientavailability or nutritional value of biomass (e.g., by modifying (e.g.,increasing, decreasing, or maintaining) the solubility of the biomassand/or changing the structure (e.g., functionalizing) of the nativematerials, and/or altering (e.g., lowering) the molecular weight and/orcrystallinity), as described above, thereby producing a more valuablefoodstuff. In some embodiments, the methods described herein can be usedto increase the nutrient availability of the biomass by breaking downcellulosic material (e.g., cellulose and/or hemicellulose) into shorterchain saccharides and/or monosaccharides. By improving the nutrientavailability of the biomass, these methods will result in a moreefficient foodstuff that can be used to maintain or improve the healthof a specific type or species of animal, promote the growth of a targetanimal (e.g., tissue gain), and/or to promote food production.

In some embodiments, a useful animal foodstuff can include partiallyprocessed biomass, e.g., biomass that has been sheared using the methodsdescribed herein. Such partially processed biomass can be more readilyhydrolyzed in the stomach of an animal.

In some embodiments, the methods described herein can be used to processbiomass to generate the materials described herein. These materials caninclude, but are not limited to, e.g., polysaccharides with a length ofgreater than 1000 saccharide units; about 1000 sugar saccharide units;about 800-900 saccharide units; about 700-800 saccharide units; about600-700 saccharide units; about 500-600 saccharide units; about 400-500saccharide units; about 300-400 saccharide units; about 200-300saccharide units; about 100-200 saccharide units; 100, 90, 80, 70, 60,50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9,8, 7, 6, 5, 4, 3, 2, and 1 saccharide units.

In some embodiments, the methods produce disaccharides (e.g., sucrose,lactose, maltose, trehalose, and cellobiose). In some embodiments, themethods produce monosaccharides (e.g., glucose (dextrose), fructose,galactose, xylose, and ribose). These shorter chain molecules will bemore easily absorbed by an animal and will thereby increase the nutrientavailability of biomass. Consequently, the methods and materialsdescribed herein can be used as foodstuffs or in the production offoodstuffs.

In some embodiments, the materials described herein can be used as afoodstuff e.g., agricultural foodstuffs and/or foodstuffs suitable foringestion by mammals, birds, and/or fish. Alternatively or in addition,the methods described herein can be used to process a raw materialsuitable for use as or in an animal foodstuff.

Materials that can be usefully processed using the methods describedherein include cellulosic and lignocellulosic materials, e.g., arableproducts, crops, grasses, plants and/or feed grains, for exampleincluding, but not limited to, plant material (e.g., forage such asalfalfa meal, hay, Bermuda coastal grass hay, sweet grass, corn plant,and soybean hay), grains (e.g., barley, corn (including organic andgenetically modified corn), oats, rice, sorghum, and wheat), plantprotein products (e.g., canola meal, cottonseed cakes and meals,safflower meal, and soybean (including organic and genetically modifiedsoybean) feed and meal), processed grain by-products (e.g., distillersproducts, brewers dried grains, corn gluten, sorghum germ cake and meal,peanut skins, and wheat bran), fruit and fruit by-products (e.g., driedcitrus pulp, apple pomace, and pectin pulp), molasses (e.g., beet,citrus, starch, and cane molasses), almond hulls, ground shells,buckwheat hulls, legumes and legume by-products, and other cropby-products. Other raw materials include, but are not limited to,alfalfa, barley, birdsfoot trefoil, brassicas (e.g., chau moellier,kale, rapeseed (canola), rutabaga (swede), and turnip), clover (e.g.,alsike clover, red clover, subterranean clover, and white clover), grass(e.g., false oat grass, fescue, Bermuda grass, brome, heath grass,meadow grass, orchard grass, ryegrass, and Timothy grass), maize (corn),millet, oats, sorghum, and soybeans. In some embodiments, the rawmaterial can be animal waste (e.g., ruminant waste) or human waste.

In some embodiments, the foodstuff contains only the materials producedusing the methods described herein. Alternatively or in addition, thefoodstuff contains additional raw materials (including raw materials nottreated using the methods described herein) and additives. Suchfoodstuffs can be formulated to meet the specific requirements of atarget animal, e.g., to maintain or improve the health of a specifictype or species of animal, to promote the growth of a target animal,tissue gain, and/or to promote food production. In some cases, afoodstuff can be formulated to meet the nutritional requirements of atarget animal for the least cost (the “least cost ration”). Methods fordetermining the formulation of a foodstuff and the least cost ration arewell known to those of skill in the art (see, for example, Pesti andMiller, Animal Feed Formulation: Economic and Computer Applications(Plant and Animal Science), Springer Publishing, Feb. 28, 1993 and worldwide web address liveinformatics.com).

Additional raw materials and additives that can be usefully combinedwith a material produced using the methods described herein include, butare not limited to, animal products (e.g., meat meal, meat meal tankage,meat and bone meal, poultry meal, animal by-product meal, dried animalblood, blood meal, feather meal, egg-shell meal, hydrolyzed wholepoultry, hydrolyzed hair, and bone marrow), animal waste, marineproducts and by-products (e.g., krill, fish parts and meal, fish residuemeal, crab parts and meal, shrimp parts and meal, fish oil, fish liverand glandular meal, and other fish by-products), dairy products (e.g.,dried cow milk, casein, whey products, and dried cheese), fats and oils(e.g., animal fat, vegetable fat or oil, and hydrolyzed fats),restaurant food waste (e.g., food waste from restaurants, bakeries, andcafeterias), and contaminated/adulterated food treated to destroypathogens. Other additives include antibiotics (e.g., tetracyclines,macrolides, fluoroquinolones, and streptogramins), flavoring, brewersoats, by-products of drug manufacture (e.g., spent mycelium andfermentation products), minerals and trace minerals (e.g., bonecharcoal, calcium carbonate, chalk rock, iron salts, magnesium salts,oyster shell flour, and sulphate), proteinated minerals (e.g.,proteinated selenium and chromium), vitamins (e.g., vitamin A, vitaminD, vitamin B₁₂, niacin, and betaine), direct fed organisms/probiotics(e.g., Aspergillus niger, Baccillus subtillis, Bifidobacterium animalis,B. bifidium, Enterococcus faecium, Aspergillus oryzae, Lactobacillusacidophilus, L. bulgaricus, L. planetarium, Streptococcus lactis, andSaccharomyces cerevisiae), prebiotics (e.g., mannan-oligosaccharides(MOS), fructo-oligosaccharides, and mixed oligo-dextran), flavors (e.g.,aloe vera gel concentrate, ginger, capsicum, and fennel), enzymes (e.g.,phytase, cellulase, lactase, lipase, pepsin, catalase, xylanase, andpectinase), acetic acid, sulfuric acid, aluminum salts, dextrans,glycerin, beeswax, sorbitol, riboflavin, preservatives (e.g., butylatedhydroxyanisole and sodium bisulfite), nutraceuticals (e.g., herbal andbotanical products), amino acids, by pass protein, urea, molasses, fattyacids, (e.g., acetic, propionic, and butyric acid) and metabolicmodifiers (e.g., somatotropins and adrenergic agonists). In some cases,the materials produced using the methods described herein can becombined or incorporated into a urea molasses mineral block (UMMB).

Foodstuffs prepared using the materials described herein can be in aform suitable for ingestion, e.g., by a target animal. In some cases thefoodstuff can be a solid. Alternatively or in addition, the foodstuffcan be in a liquid form, e.g., the foodstuff can be in a liquidsuspension or solution in a suitable solvent. Exemplary forms include,but are not limited to solids such as powders, tablets, mineral blocks,pellets, biscuit, and mixtures of an unprocessed raw material (e.g.,grass) and a material processed using the methods described herein.

In some embodiments, the materials described herein can be incorporated(e.g., mixed) into a foodstuff by a farmer, e.g., for local use and/orsmall scale distribution. In such cases, the materials described hereincan be provided to the farmer in a packaged form, e.g., in a form thatis suitable for incorporation into a foodstuff. Alternatively or inaddition, the materials described herein can be incorporated (e.g.,mixed) into a foodstuff by a foodstuff manufacturer, e.g., for largescale distribution. In such cases, the materials described herein can beprovided to the foodstuff manufacturer in a form that is suitable forincorporation into a foodstuff. Alternatively or in addition, thematerials described herein can be prepared from a raw material at thesite at which the foodstuff is prepared.

In some embodiments, the materials described herein can be distributedalone and ingested by an animal in the absence of any additional rawmaterials and/or additives.

In some embodiments, the materials will require post-processing prior touse as food. For example, a dryer can be used to remove moisture fromthe intermediate fermentation products to facilitate storage, handling,and shelf life. Additionally or alternatively, the materials can beground to a fine particle size in a stainless-steel mill to produce aflour-like substance.

Typically, biomass based foodstuffs are usefully fed only to ruminantsthat are capable of at least partially digesting cellulose. As thepresent disclosure provides materials in which the cellulosic materialhas been broken down into shorter chain sugars, these materials can alsobe used as a viable foodstuff for animals that are incapable ofcellulose or hemicellulose digestion. Therefore, foodstuffs preparedusing the materials and methods described herein can be usefully fed toanimals including, but not limited to, food production animals, zooanimals, and laboratory animals, and/or domestic animals. The foodstuffscan also be used in agriculture and aquaculture. In addition, becausefoodstuffs prepared using the materials described herein have a highernutrient availability, less foodstuff will be required by the animal toreceive the same amount of energy, which will reduce the overall cost ofthe foodstuff. Alternatively, animals will be able to consume moreenergy, which will result in higher growth rates, tissue gain, milkproduction, and egg production.

In some embodiments, the materials described herein can be usefully fedto ruminants (e.g., cattle, goats, sheep, horses, elk, bison, deer,camels, alpacas, llamas, giraffes, yaks, water buffalo, wildebeest, andantelope), poultry, pigs, boars, birds, cats, dogs, and fish.

In some embodiments, distillers grains and solubles can be convertedinto a valuable byproduct of the distillation-dehydration process. Afterthe distillation-dehydration process, distillers grains and solubles canbe dried to improve the ability to store and handle the material. Theresulting dried distillers grains (DDG) and solubles is low in starch,high in fat, high in protein, high in fiber, and high in phosphorous.Thus, for example, DDG can be valuable as a source of animal feed (e.g.,as a feed source for dairy cattle). DDG can be subsequently combinedwith nutritional additives to meet specific dietary requirements ofspecific categories of animals (e.g., balancing digestible lysine andphosphorus for swine diets). Alternatively or in addition, biomassprocessed using the methods described herein can be combined with DDG.The ratio of processed biomass to DDG can be optimized to meet the needsof target animals.

In some embodiments, oils obtained from biomass using the methodsdescribed herein can be used in animal feed, e.g., as a pet foodadditive.

In some embodiments, as obtained from biomass using the methodsdescribed herein can be used in animal feed.

Human Foodstuffs

As described above, humans are typically less able to digest celluloseand cellulosic material. Biomass is an abundantly available material,however, that could serve as a novel foodstuff for human consumption. Inorder for a biomass material (e.g., a material containing cellulose) tobe useful as a human foodstuff, however, the nutrient availability ofthe biomass would have to be increased by (1) increasing the solubilityof the biomass; (2) changing the structure (e.g., functionalizing) ofthe native materials; (3) altering (e.g., lowering) the molecular weightand/or crystallinity relative to a native material; and/or (4) breakingdown cellulosic material into smaller saccharides, for example,saccharides with a length of greater than 1000 saccharide units; about1000 sugar saccharide units; about 800-900 saccharide units; about700-800 saccharide units; about 600-700 saccharide units; about 500-600saccharide units; about 400-500 saccharide units; about 300-400saccharide units; about 200-300 saccharide units; about 100-200saccharide units; 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 19,18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, and 1saccharide units. Such materials will have an increased nutrientavailability (as described above), e.g., in humans, and will be usefulas a human foodstuff. In general, a useful human foodstuff should, e.g.,provide a usable and accessible energy and nutrient source to the humanto, e.g., maintain or improve the health of a human, and/or promote thegrowth of a human (e.g., tissue gain). The methods described herein canbe used to produce such a useful human foodstuff, e.g., from abiomass-based material.

In some embodiments, the materials will require processing prior to useas food. For example, a dryer can be used to remove moisture from theintermediate fermentation products to facilitate storage, handling, andshelf life. Additionally or alternatively, the materials can be groundto a fine particle size in a stainless-steel mill to produce aflour-like substance.

Such foodstuffs can include, but are not limited to, for example, energysupplements (e.g., powders and liquids). Alternatively or in addition,the materials described herein can be combined with a first food toincrease the nutritional value of the first food. For example, thefoodstuffs described herein can be combined with a low energy food toincrease the energy of the food.

Alternatively or in addition, the materials described herein can be usedto increase the sweetness of the food, e.g., as a sweetening agent, aswell as the nutritional value of the food. In such cases, it can bedesirable to obtain one or more specific sugars (e.g., a monosaccharide,a disaccharide, a oligosaccharide, and/or a polysaccharide) from thematerials, e.g., by isolating the one or more specific sugars from thematerials. Methods for isolating sugars are known in the art.

In some embodiments, the materials described herein can be used as a lowcost material for food production. For example, the materials can besupplied to bakeries for use in bread and/or confectionary, and to foodmanufacturers to be used as a filler, e.g., to increase the volumeand/or nutritional value of a food.

In some embodiments, the materials can further serve as a source offiber for human consumption. In such cases, the methods used to breakdown the cellulolytic material will be configured to provide a lesscomplete reduction in molecular weight, e.g., the methods will result inmaterials containing some cellulose and/or result in longer chain lengthpolysaccharides that are not easily absorbed by humans. Such materialscan be fed to a human in the form of a solid (e.g., a tablet or agranular powder) or a liquid (e.g., a solution, gel, colloid, orsuspension).

In some embodiments, the materials described herein can be fed to ahuman alone or in the combination with a second food that is suitablefor human consumption. Such foods include, but are not limited to,breads, dairy products, meats, fish, cereals, fruits, vegetables, beans(e.g., soy), and gums. In some embodiments, the materials describedherein can be combined with protein, fats, carbohydrates, minerals,pharmaceuticals, and vitamins.

Proteins

In some embodiments, the methods described herein can be used to obtain(e.g., extract, isolate, and/or purify proteins (e.g., polypeptides,peptides, and amino acids) from biomass. Such proteins (e.g.,polypeptides, peptides, and amino acids) can be used, e.g., alone or incombination with one or more of the materials and biomass componentsobtained using the methods described herein, in the food industry (e.g.,as additives, supplements, and/or fillers), in the cosmetic industry(e.g., in the compounding of cosmetics), and/or in agriculture (e.g., asfoodstuffs or to feed or maintain crops) or aquaculture.

In some embodiments, the methods described herein can be used to obtainproteins (e.g., polypeptides, peptides, and amino acids) from e.g., okraseed, Lipinus mutabilis, nuts (e.g., macadamia nuts), Jessenia bataua,Oenocarpus, Stokesia laevis, Veronia galamensis, and Apodantheraundulate.

Fats, Oils, and Lipids

Fats consist of a wide group of compounds that are generally soluble inorganic solvents and largely insoluble in water. Fats that are solid atroom temperature. Fats that are liquid at room temperature are typicallyreferred to as oils. The term lipids typically refers to solid andliquid fats. As used herein, the terms fats, oils, and lipids include,but are not limited to, edible oils, industrial oils, and thosematerials having an ester, e.g., triglyceride and/or hydrocarbon.

In some embodiments, the methods described herein can be used to obtain(e.g., extract, isolate, and/or purify) fats (e.g., lipids, and fattyacids) from biomass. Such fats (e.g., lipids, and fatty acids) can beused, e.g., alone or in combination with one or more of the materialsand biomass components obtained using the methods described herein, inthe food industry (e.g., as additives, supplements, and/or fillers), inthe cosmetic industry (e.g., in the compounding of cosmetics), and/or inagriculture (e.g., as foodstuffs).

In some embodiments, the methods described herein can be used to obtain(e.g., extract, isolate, and/or purify) oils from biomass. Such oils canbe used, e.g., alone or in combination with one or more of the materialsand biomass components obtained using the methods described herein, inthe food industry (e.g., as additives, supplements, and/or fillers), inthe cosmetic industry (e.g., in the compounding of cosmetics), inagriculture (e.g., as foodstuffs), as biofuels, drying oils (e.g., inpaints), and pet food additives.

In some embodiments, the methods described herein can be used to obtainfats, oils, and/or lipids from e.g., sunflower, okra seed, buffalo gourd(Cucurbita foetidissima), Lipinus mutabilis, nuts (e.g., macadamianuts), Jessenia bataua, Oenocarpus, Crambe abyssinica (Crambe),Monoecious jojoba (jojoba), Cruciferae sp. (e.g., Brassica juncea, B.carinata, B. napas (common rapeseed), and B. campestris), Stokesialaevis, Veronia galamensis, and Apodanthera undulate.

Carbohydrates and Sugars

In some embodiments, the methods described herein can be used to obtain(e.g., extract, isolate, and/or purify) carbohydrates and/or sugars frombiomass. Such carbohydrates and sugars can be used, e.g., alone or incombination with one or more of the materials and biomass componentsobtained using the methods described herein, e.g., in the food industry(e.g., as additives, supplements, syrups, and/or fillers), in thecosmetic industry (e.g., in the compounding of cosmetics), and/or inagriculture (e.g., as foodstuffs).

Vitamins

In some embodiments, the methods described herein can be used to obtain(e.g., extract, isolate, and/or purify) vitamins from biomass. Suchvitamins can be used, e.g., alone or in combination with one or more ofthe materials and biomass components obtained using the methodsdescribed herein, e.g., in the food industry (e.g., as additives, andsupplements), in the healthcare industry, in the cosmetic industry(e.g., in the compounding of cosmetics), and/or in agriculture.

Minerals

In some embodiments, the methods described herein can be used to obtain(e.g., extract, isolate, and/or purify) minerals from biomass. Suchminerals can be used, e.g., alone or in combination with one or more ofthe materials and biomass components obtained using the methodsdescribed herein, e.g., in the food industry (e.g., as additives, andsupplements), in the healthcare industry, in the cosmetic industry(e.g., in the compounding of cosmetics), and/or in agriculture.

Ash

In some embodiments, the methods described herein can be used to obtain(e.g., extract, isolate, and/or purify) ash from biomass. Such ash canbe used, e.g., alone or in combination with one or more of the materialsand biomass components obtained using the methods described herein,e.g., in the food industry (e.g., as an additive, supplement, and/orfiller).

Pharmaceuticals

Over 120 currently available pharmaceutical products are plant-derived.As the methods described herein are useful for processing cellulolyticmaterial, these methods can be useful in the isolation, purification,and/or production of plant-based pharmaceuticals.

In some embodiments, the materials described herein can have medicinalproperties. For example, the methods described herein can result in theproduction of a material with novel medicinal properties (e.g., notpresent in the native material). Alternatively or in addition, themethods described herein can result in the production of a material withincreased medicinal properties (e.g., a greater medicinal property thanthat of the native material).

In some embodiments, the methods described herein can be used to modify(e.g., increase, decrease, or maintain) the solubility of a material,e.g., a material with medicinal properties. Such a material can be moreeasily administered and/or absorbed, e.g., by a human and/or animal thana native material.

In some embodiments, the methods described herein can be used tofunctionalize (e.g., alter the structure, expose a reactive side chain,and/or modify the charge) a material with medicinal properties. Suchmaterials can have, e.g., altered reactivity, altered charge, and/oraltered solubility.

In some embodiments, the methods described herein can be used to modifythe molecular structure of a material, e.g., a material with medicinalproperties. Such materials can have altered (e.g., increased ordecreased) average molecular weights, average crystallinity, surfacearea, and/or porosity. Such materials can have, e.g., alteredreactivity, altered charge, altered solubility.

In some embodiments, the methods described herein can be used as highefficiency processing methods, e.g., to obtain plant-basedpharmaceuticals from a cellulosic raw material such as plants. In someembodiments, the methods described herein can be used to increase thepharmaceutical activity of a plant-based pharmaceutical. For example, insome embodiments, the methods described herein can be applied to plantsand/or herbs with medicinal properties. For example, sonication canstimulate bioactivity and/or bioavailability of the medicinal componentsof plants and/or herbs with medicinal properties. Additionally oralternatively, irradiation can stimulate bioactivity and/orbioavailability of the medicinal components of plants and/or herbs withmedicinal properties.

In some embodiments, the methods described herein can be used toincrease the solubility of a plant and/or herbal material. Alternativelyor in addition, the methods described herein can be used to reduce thetoxicity of a plant and/or herbal material without reducing themedicinal properties of the plant and/or herb. In some embodiments, themethods described herein are useful for isolating and/or purifyingpharmaceutical compounds from plant material (which without being boundby theory, is possible due to the more efficient break down ofcellulosic material) as the methods disrupt, alter, modify, orrestructure cellulose, e.g., present in the leaves of plant material.Desired compounds released using the methods described herein can thenbe isolated from the undesired material, whereas less efficient methodscan not allow the release of the desired material from undesiredmaterial. Inevitably, therefore, less efficient methods will result inthe carry over of undesired material, which can lower the efficacy ofthe desired (e.g., pharmaceutical compound) and/or be associated withpotentially toxic side effects. The methods described herein can,therefore, be used to generate highly purified forms of potentiallypharmaceutical compounds, e.g., free of undesirable plant material, thatare not obtainable using current practices. These highly purifiedcompounds can be more efficacious then less purified forms of the samecompounds. In some embodiments, the increased efficacy attainable usingthe methods described herein can allow reduced dosing. In turn, thisreduction in the amount of material administered to a subject can reduceassociated toxicity. Alternatively or in addition, the removal ofsurplus or undesirable plant material can help reduce or eliminate anytoxicity associated with a plant based compound that has not beenprocessed using the methods described herein.

Examples of plants and/or plant material that can be usefully treatedusing the methods described herein include, for example, sonication andirradiation can be combined in the pretreatment of willow bark tostimulate the isolation, purification, and/or production of salicin.Alternatively or in addition, the methods described herein can be usedto process plant material comprising comfrey plants to facilitate theisolation, purification, and/or production of allantoin. Alternativelyor in addition, the methods described herein can be used to facilitatethe isolation, purification, and/or production benzoin. Alternatively orin addition, the methods described herein can be used to process plantmaterial comprising camphor basil to facilitate the isolation,purification, and/or production of camphor. Alternatively or inaddition, the methods described herein can be used to process plantmaterial comprising plants in the genus Ephedra to facilitate theisolation, purification, and/or production of ephedrine. Alternativelyor in addition, the methods described herein can be used to processplant material comprising Duboisia myoporoides R. Br. (Australian corktree) to facilitate the isolation, purification, and/or production ofatropine. In some embodiments, the atropine obtained using the methodsdescribed herein will have an increased anticholinergic effect.Alternatively or in addition, the methods described herein can be usedto process plant material comprising Mucuna deeringiana (velvet bean) tofacilitate the isolation, purification, and/or production of L-dopa. Insome embodiments, the L-dopa obtained using the methods described hereinwill have an increased antiparkinsonian effect. Alternatively or inaddition, the methods described herein can be used to process plantmaterial comprising Physostigma venenosum Balf: (ordeal bean) tofacilitate the isolation, purification, and/or production ofphysostigmine. In some embodiments, the physostigmine obtained using themethods described herein will have an increased anticholinesteraseeffect. Examples of other plant-based pharmaceuticals in which themethods described herein can be used to process plant material tofacilitate the isolation, purification, and/or production of include,but are not limited to, bromelain, chymopapain, cocaine, deserpidine,emetine, hyoscyamine, kawaina, monocrotaline, ouabain, papain,pilocarpine, quinidine, quinine, rescinnami, reserpine, scopolamine,tubocurarine, vinblastine, yohimbine, caprylic-acid, cineole, citricacid, codeine, cresol, guaiacol, lecithin, menthol, phenolpseudephedrine, sorbitol, and tartaric acid.

In some embodiments, the methods described herein can be used to processherbs, e.g., medicinal herbs, including, but not limited to, basil,lemon grass, parsley, peppermint, and celery. Additional medicinal herbsthat can be processed using the methods described herein can be found atworld wide web address altnature.com.

An emerging technology is the production of pharmaceuticals in plants.Pharmaceutical produced using plants, which are commonly referred to asplant made pharmaceuticals (PMPs), include pharmaceutical compounds andvaccines. Typically, PMPs are expressed in the leaves of the respectiveplants. Clearly, therefore, the methods described herein can be usefulfor processing plant material comprising PMPs to facilitate theisolation, purification, and/or production of the PMPs.

Additional exemplary medicinal plants that can be treated using themethods described herein can be found e.g., at world wide web addressnps.gov/plants/MEDICINAL/plants.htm.

In some embodiments, material that has been processed using the methodsdescribed herein can be combined with a pharmaceutical excipient, e.g.,for administration to a subject. Exemplary excipients that can be usedinclude buffers (for example, citrate buffer, phosphate buffer, acetatebuffer, and bicarbonate buffer), amino acids, urea, alcohols, ascorbicacid, phospholipids, polypeptides (for example, serum albumin), EDTA,sodium chloride, liposomes, mannitol, sorbitol, water, and glycerol.Dosage forms can be formulated to be suitable for any standard route ofadministration. For example, administration can be parenteral,intravenous, subcutaneous, or oral or any route of administrationapproved by the Federal Drug Administration (see world wide web addressfda.gov/cder/dsm/DRG/drg00301.htm).

Nutriceuticals and Nutraceuticals

Foods with a medical health benefit, including the prevention and/ortreatment of disease, are referred to as nutraceuticals ornutriceuticals. For example, nutraceuticals and nutriceuticals arenaturally occurring or artificially generated nutritional supplementscapable of promoting a healthy lifestyle, for example, by reducingdisease related symptoms, reducing the incidence or severity of disease,and promoting long-term health.

In some embodiments, the methods described herein can be used togenerate combinations of monosaccharides, disaccharides,oligosaccharides, and/or polysaccharides that are capable of promoting ahealthy lifestyle. In some embodiments, the methods described herein canbe used to generate a material that is useful for promoting weight lossin a human. For example, the material can have low nutrient availabilitywith low digestibility, e.g., a fibrous material. Such materials couldbe used as a dietary supplement, e.g., to suppress hunger and/or topromote satiety. Consuming such materials would allow a subject to avoidconsuming high nutrient availability and/or highly digestible foods andthus would facilitate weight loss in the individual.

In some embodiments, the materials described herein can be supplementedwith one or more nutritional supplements that are capable of promoting ahealthy lifestyle. In such cases, the materials described herein caneither enhance the activity of the one or more nutritional supplementsand/or enhance the solubility and/or pharmacokinetics of the one or morenutritional supplements. Exemplary nutritional supplements that can becombined with the materials described herein include, but are notlimited to, for example, silica, silicon, boron, potassium, iodine,beta-carotene, lycopene, insoluble fiber, monosaturated fatty acids,omega-3 fatty acid, flavonols, sulforaphane, phenols (e.g., caffeic acidand ferulic acid), plant stanols and sterols (including esters thereof),polyols (e.g., sugar alcohols), prebiotics and probiotics (e.g.,Lactobacilli and bifidobacteria), phytoestrogens (e.g., isoflavones suchas daidzein and genistein), proanthocyanidins, soy protein, sulfides andthiols (e.g., dithiolthiones), vitamins (e.g., vitamin A, vitamin B₁,vitamin B₂, vitamin B₃, vitamin B₅, vitamin B₆, vitamin B₇, vitamin B₁₂,vitamin C, vitamin D, vitamin E, vitamin K, including combinationsthereof) minerals (e.g., iron, calcium, magnesium, manganese,phosphorus, potassium, zinc, trace minerals, chromium, selenium,including combinations thereof), and folic acid.

Pharmaceutical Dosage Forms and Drug Delivery Compositions

Drug substances are seldom administered alone, but rather as part of aformulation in combination with one or more non-medical agents thatserve varied and often specialized pharmaceutical functions.Pharmaceutics is the science of dosage form design, e.g., formulating adrug into a dosage form suitable for administration to a subject. Thesenon-medical agents, referred to as pharmaceutic or pharmaceuticalingredients, can be formulated to solubilize, suspend, thicken, dilute,emulsify, stabilize, preserve, color, flavor, and fashion medicinalagents into efficacious and appealing dosage forms. Such dosage formscan be unique in their physical and pharmaceutical characteristics. Thedrug and pharmaceutic ingredients will typically be compatible with eachother to produce a drug product that is stable, efficacious, attractive,easy to administer, and safe. The product should be manufactured underappropriate measures of quality control and packaged in containers thatcontribute to promote stability. Methods describing the preparation ofspecific dosage forms are well known in the art and can be found in, forexample, Ansel et al., Pharmaceutical Dosage Forms and Drug DeliverySystems, Seventh Edition, Lippincott, Williams, & Wilkins and,“Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., MackPublishing Co., Easton, Pa., 1990.

In some embodiments, the pretreated materials described herein can beused as pharmaceutic ingredients e.g., inactive ingredients. Forexample, the materials described herein can be used, e.g., formulated,to solubilize, suspend, thicken, dilute, emulsify, stabilize, preserve,color, flavor, and fashion medicinal agents into efficacious, palatable,and appealing dosage forms. In such cases, the materials describedherein can be mixed with a drug and/or conjugated to a drug such thatthe solubility, concentration, viscosity, emulsion stability, shelflife, color, and flavor of the drug is increased or decreased.

In some embodiments, the methods described herein can be used to modify(e.g., increase, decrease, or maintain) the solubility of a material.Such materials can be used to facilitate the administration of a drug toa subject. For example, certain of the new pretreated materials areexceptionally soluble in liquids, such as water, and can be used, whenmixed with active ingredients to form a pharmaceutical composition, toallow the inert ingredients to be easily dissolved in liquids.

Alternatively or in addition, the materials described herein can be usedto delay, control, or modify the release of a drug once the drug hasbeen administered to a subject. In such cases, the materials describedherein can be used in solid dosage forms and/or controlled-release drugdelivery systems; semi-solid and/or transdermal systems; pharmaceuticalinserts; liquid dosage forms; sterile dosage forms and delivery systems;and novel and advanced dosage forms, delivery systems and devices. Forexample, the materials described herein can be formulated, e.g., in theform of a tablet, a capsule (e.g., a hard capsule, a soft capsule, or amicrocapsule), a suppository, an injectable solution or suspension (aparenteral), a cream, a ointment, an ophthalmic solution or suspension,an ear drop solution or suspension, an inhalable solution or suspension,a nasal spray, a transdermal patch, an emulsion, a ointment, a cream, agel, a suspension, a dispersion, a solution (e.g., an intravenoussolution), an implant, a coating for an implant, a lotion, a pill, agel, a powder, and a paste. In some embodiments, the materials describedherein can be combined with a radiopharmaceutical.

In some embodiments, the methods described herein can be used togenerate a material that can be conjugated to a biological agent and/ora pharmaceutical agent. Such conjugates can be used to facilitateadministration of the agent and increase the pharmaceutical propertiesof the agent.

The formulations and routes of administration can be tailored to thedisease or disorder being treated, and for the specific human beingtreated. When using the materials described herein as pharmaceuticingredients, it can be necessary to determine the optimal formulationand dosage type. For example, various initial formulations can bedeveloped and examined for desired features (e.g., drug release profile,bioavailabilty, and clinical effectiveness) and for pilot plant studiesand production scale up. The formulation that best meets the goals forthe product (e.g., drug release profile, bioavailabilty, and clinicaleffectiveness) can then be selected as the master formula. Eachsubsequent batch of product can then be prepared to meet thespecifications of the master formula. For example, if the product is forsystemic use and oral administration is desired, tablets and/or capsulesare usually prepared. The age of the intended patient can also beconsidered when selecting a dosage form. For example, for infants andchildren younger than five years of age, pharmaceutical liquids ratherthan solids are preferred for oral administration. In addition, thephysical characteristics of the drug or drugs to be formulated with thepharmaceutic ingredients must be understood prior to dosage formdevelopment.

Pharmaceutical compositions containing one or more of the compoundsdescribed herein will be formulated according to the intended method ofadministration.

In some cases, the nature of the dosage form is dependent on the mode ofadministration and can readily be determined by one of ordinary skill inthe art. In some embodiments, the dosage form is sterile orsterilizable. In particular, the materials described herein are oftensterile when pretreated with radiation as described herein.

In some embodiments, the dosage forms can contain carriers orexcipients, many of which are known to skilled artisans. Exemplaryexcipients that can be used include buffers (for example, citratebuffer, phosphate buffer, acetate buffer, and bicarbonate buffer), aminoacids, urea, alcohols, ascorbic acid, phospholipids, polypeptides (forexample, serum albumin), EDTA, sodium chloride, liposomes, mannitol,sorbitol, water, and glycerol. Dosage forms can be formulated to besuitable for any standard route of administration. For example,administration can be parenteral, intravenous, subcutaneous, or oral orany route of administration approved by the Federal Drug Administration(see world wide web address fda.gov/cder/dsm/DRG/drg00301.htm).

In addition to the formulations described previously, the compositionscan also be formulated as a depot preparation. Such long-actingformulations can be administered, e.g., by implantation (e.g.,subcutaneously). Thus, for example, the compositions can be formulatedwith suitable polymeric or hydrophobic materials (for example, as anemulsion in an acceptable oil) or ion exchange resins, or as sparinglysoluble derivatives, for example, as a sparingly soluble salt.

Pharmaceutical compositions formulated for systemic oral administrationcan take the form of tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents (forexample, pregelatinized maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (for example, lactose,microcrystalline cellulose or calcium hydrogen phosphate); lubricants(for example, magnesium stearate, talc, or silica); disintegrants (forexample, potato starch or sodium starch glycolate); or wetting agents(for example, sodium lauryl sulphate). Many of the functions of thesebinding agents, fillers, lubricants, and disintegrants can be served bythe pretreated materials described herein.

The tablets can be coated by methods well known in the art. Liquidpreparations for oral administration can take the form of, for example,solutions, syrups or suspensions, or they can be presented as a dryproduct for reconstitution with water or other suitable vehicle beforeuse. Such liquid preparations can be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (forexample, sorbitol syrup, cellulose derivatives or hydrogenated ediblefats); emulsifying agents (for example, lecithin or acacia); non-aqueousvehicles (for example, almond oil, oily esters, ethyl alcohol orfractionated vegetable oils); and preservatives (for example, methyl orpropyl-p-hydroxybenzoates or sorbic acid). The preparations can alsocontain buffer salts, flavoring, coloring and sweetening agents asappropriate. Preparations for oral administration can be suitablyformulated to give controlled release of the active compound.

In some embodiments, the materials described herein can be used aspharmaceutic ingredients for use in topical iontophoresis,phonophoresis, rapidly dissolving tablets, lyophilized foam, anintravaginal drug delivery system, a vaginal insert, a urethral insertor suppository, an implantable drug delivery pump, an external drugdelivery pump, and a liposome.

Hydrogels

In some embodiments, the materials described herein can be used in theformulation of a hydrogel. Hydrogels are three-dimensional networks ofhydrophilic polymer chains that are crosslinked through either chemicalor physical bonding and are water insoluble and are typicallysuperabsorbent (e.g., can contain over 99% water) and permit gas andnutrient exchange.

In some embodiments, the materials described herein can be used togenerate a hydrogel. For example, monosaccharides, oligosaccharides, andpolysaccharides contained in the materials described herein can be usedto generate a hydrogel. Alternatively or in addition, the materialsdescribed herein can be used to generate a hydrogel in combination withother materials such as hyaluronan, gelatin, cellulose, silicone, andone or more components of the extracellular matrix (ECM).

In some embodiments, hydrogels containing the materials described hereincan be cross-linked (e.g., chemically cross-linked) and/or oxidized.Alternatively or in addition, hydrogels containing the materialsdescribed herein can be cross-linked using low-level irradiation. Dosesof low-irradiation that can be used to cross-link the materialsdescribed herein include, but are not limited to, for example, 0.1 Mradto 10 Mrad. Alternatively or in addition, hydrogels containing thematerials described herein can be cross-linked using a combination ofchemical cross-linking, low-level irradiation, and oxidation.

In some embodiments, the methods described herein can be used to modify(e.g., increase) the average molecular weight of the biomass materialsdescribed herein. For example, the methods described herein can be usedto increase the average molecular weight of a biomass material by, e.g.,10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, or as much as 500%.

In some embodiments, the methods described herein can be used to modify(increase or decrease) the Poisson's ratio of a hydrogel.

In some embodiments, hydrogels generated using the materials describedherein can include one or more one or more biological cells and/or oneor more bioactive agent such as a pharmaceutical agent or a component ofthe ECM. Candidate pharmaceutical agents, could include but are limitedto, a therapeutic antibody, an analgesic, an anesthetic, an antiviralagent, an anti-inflammatory agent, an RNA that mediates RNAinterference, a microRNA, an aptamer, a peptide or peptidomimetic, animmunosuppressant, hypoxyapatite, or bioglass.

Hydrogels containing the materials described herein can be used asbiodegradable or non-biodegradable implantable (e.g., subdermalimplantable) three-dimensional scaffolds, e.g., in wound healing andtissue engineering, implantable disc replacements, drug deliveryvehicles (e.g., slow release drug delivery vehicles), on wound dressing,contact lenses, and as superabsorbant materials (e.g., in diapers).

Hydrogels containing the materials described herein can also be combinedwith medical devices for the treatment of both external and internalwounds. The hydrogels can be applied to bandages for dressing externalwounds, such as chronic non-healing wounds, or used as subdermalimplants. Alternatively, the present hydrogels can be used in organtransplantation, such as live donor liver transplantation, to encouragetissue regeneration. The hydrogels can be adapted to individual tissuetypes by equilibrating the water content, biodegradation kinetics, andPoisson's ratio with those of the target tissue to be repaired.

Methods for making hydrogels are well known in the art and can be found,for example, in U.S. 2006/0276608.

Absorbent Materials

In some embodiments, the methods described herein can be used togenerate absorbent materials. For example, in some embodiments, biomasscan be processed using one or more of the pretreatment methods describedherein. Such materials can have, e.g., modified (increased, decreased,maintained) solubility, porosity, surface area, average molecularweight, functionalization (e.g., an increased number of hydrophilicgroups). Alternatively or in addition, these materials can be chemicallytreated to enhance a specific absorption property. For example, thematerials can be treated with silanes to render them lipophilic. Thesematerial can have the ability to absorb 1, 2, 5, 10, 20, 50, 100, 500,and 1000 times more fluid than native materials and/or 1, 2, 5, 10, 20,50, 100, 500, and 1000 times the materials own weight. In someembodiments, these materials can be used to adhere (e.g., selectively)to one or more materials (e.g., biological materials in blood or plasma,toxins, pollutants, waste materials, inorganic chemicals, and organicchemicals), e.g., in a solution or in a dry medium

In some embodiments, the materials described herein can be used asabsorbent materials, e.g., for use as animal litter, e.g., for small andlarge animals, and animal bedding. Methods for making animal litter arewell known in the art (see e.g., U.S. Pat. No. 5,352,780).

In some embodiments, the absorbent animal litter will additionallyinclude a scented or fragrant material and/or an odor eliminatingmaterial as are known in the art.

In some embodiments, the materials described herein can be used toabsorb chemical spills, e.g., by applying the materials to a spill.

In some embodiments, the materials described herein can be used incombination with a filter, e.g., a medical filter or a non-medicalfilter.

The materials described herein will provide useful absorbent materialsdue to the high surface area, the high absorbency, the high swellingproperties, and the high porosity of the materials described herein.

Pollution Control

In some embodiments, the absorbent materials described herein can beused for pollution control. When used for such applications, theabsorbent materials can be used in the form of a solid, liquid, or gas.For example, the materials described herein can be used to absorb oiland/or for clean up of environmental pollution, for example, in water,in the air, and/or on land. The materials described herein can also beused for waste water treatment (e.g., factory waste and sewagetreatment), and for water purification.

In some embodiments, the absorbent materials described herein can beused in combination with biologic agents (microorganisms, fungi, greenplants or their enzymes) or chemicals to facilitate removal,inactivation, or neutralization of the pollutant from the environment,e.g., using bioremediation.

In some embodiments, the absorbent materials described herein candegrade (e.g., biodegrade). Such a process can be controlled to achievea desired degradation rate. In some embodiments, the absorbent materialsdescribed herein can be resistant to degradation. In some instances,these absorbent materials can be associated with a structure or carriersuch as netting, a membrane, a flotation device, a bag, a shell, afilter, a casing, or a biodegradable substance. Optionally, thestructure or carrier itself can be made of the materials describedherein.

Air Purification

In some embodiments, biomass processed using the methods describedherein can carry a charge (e.g., a positive or negative charge) or canbe neutral. In some embodiments, charged (e.g., positively or negativelycharged) materials can be used for the removal of contaminants (e.g.,microorganisms, spores, mild spores, dust, pollen, allergens, smokeparticles, and dust mite feces) from air. In some embodiments, charged(e.g., positively or negatively charged) materials can be used to trapcontaminants. Alternatively or in addition, charged (e.g., positively ornegatively charged) materials can be used to eliminate contaminants. Forexample, in some embodiments, the methods described herein can be usedto increase the cationic value of a material. In general, cationiccompounds have antimicrobial activity. In some cases, charged (e.g.,positively or negatively charged) materials can be combined withphenolics, pharmaceuticals, and/or toxins (e.g., listed herein) for theelimination of microorganisms and/or spores.

In some embodiments, charged (e.g., positively or negatively charged)materials can be used in conjunction with a device such as an airpurification device. For example, charged (e.g., positively ornegatively charged) materials can be mobilized on a surface within anair purification device, e.g., a filter (e.g., a fibrous filter, and/ora fibrous filter n mat form). Alternatively or in addition, charged(e.g., positively or negatively charged) materials can be present in theform of a gas and/or vapor within an air purification device.Alternatively or in addition, charged (e.g., positively or negativelycharged) materials can be used in an air handling system (e.g., an airconditioning unit), e.g., within a closed environment such as within avehicle (e.g., a car, bus, airplane, and train carriage), a room, anoffice, or a building. For example, charged (e.g., positively ornegatively charged) materials can be used can be mobilized on a surfacewithin an air handling system, e.g., a filter. Alternatively or inaddition, charged (e.g., positively or negatively charged) materials canbe present in the form of a gas and/or vapor within an air handlingsystem. Alternatively or in addition, charged (e.g., positively ornegatively charged) materials can be used more locally. In such cases,charged (e.g., positively or negatively charged) materials can becontained and dispensed from a container, e.g., a pressurized canisteror a non-pressurized container with a pump. Alternatively or inaddition, charged (e.g., positively or negatively charged) materials canbe used in a slow release system, e.g., wherein charged (e.g.,positively or negatively charged) materials are released into the airover a period of time. Such slow release systems are known in the artand are commercially available. In some embodiments, such slow releasesystems can use heat (e.g., generated using electricity) to promoterelease of the charged (e.g., positively or negatively charged)materials.

In some embodiments, charged (e.g., positively or negatively charged)materials can be used in conjunction with an air filter.

In some embodiments, charged (e.g., positively or negatively charged)materials can be used in a device designed to filter the air inhaledand/or exhaled by a human (e.g., masks, a filtration helmets, and/orfiltration suits). In some embodiments, such devices can be used toreduce the inhalation of one or more potential pollutants by a human.Alternatively or in addition, such devices can be used to reduce theexhalation of one or more potential pollutants by a human.

In some embodiments, the methods described herein can be used togenerate materials useful as aromatics. Such aromatics can be combinedwith any of the products and co-products described herein. Alternativelyor in addition, these aromatics can be used to alter the scent orfragrance of a material (e.g., a solid or liquid) and/or air. In suchcases, aromatics can be used in combination with, e.g., candles,perfumes, detergents, soaps, gels, sprays, and air fresheners. Exemplaryaromatics than can be obtained from biomass include, e.g., lignin andbio-aromatics.

Food Preservation

In some embodiments, the methods described herein can be used togenerate materials useful for food preservation, or that can be used infood preservation. In such cases, suitable materials can be in the formof a gas, a vapor, a liquid, and/or a solid. In some embodiments,materials (e.g., charged materials) can be used to trap contaminants.Alternatively or in addition, materials (e.g., charged materials) can beused to eliminate contaminants. In some cases, materials (e.g., chargedmaterials) can be combined with phenolics and/or toxins for theelimination of microorganisms and/or spores. For example, materials(e.g., charged materials) can be used for the removal of contaminants(e.g., microorganisms, spores, and mild spores) from an area surroundingfood items to prevent, limit, or reduce spoilage of food items. Forexample, materials (e.g., charged materials) can be present within acontainer transporting food items. Alternatively or in addition,materials (e.g., charged materials) can be present in a container (e.g.,a package or bag) intended for storage of a food item. Such items can besold with the materials (e.g., charged materials) can already present,or materials (e.g., charged materials) can be added upon adding a fooditem to the container. Alternatively or in addition, materials (e.g.,charged materials) can be present within a cold storage area such as afridge and/or a freezer.

Herbicides and Pesticides

In some embodiments, the methods described herein can be used togenerate toxins (e.g., natural toxins) including, but not limited to,herbicides and pesticides. Such materials include, for example, lectins,glycoalkaloids, patulin, algal toxins, paralytic shellfish poison (PSP),amnesiac shellfish poisons (ASP), diarrhetic shellfish poison (DSP),vitamin A, and mycotoxins.

Fertilizer

In some embodiments, the methods described herein can be used togenerate materials that can be used as fertilizer. Biomass is rich innutrients and is currently used as fertilizer, however, native materialhas low solubility and is only useful as a fertilizer once partially orfully decomposed, both of which can take substantial amounts of time,require some tending, and require provision of storage space whiledecomposition takes place. This generally limits the use of biomass asfertilizer.

In some embodiments, the methods described herein can be used to modifybiomass into materials with, e.g., modified (e.g., increased) solubilitythat can be used as fertilizers. Such materials can be distributed overan area in need of fertilization and will be solubilized upon contactwith a solution (e.g., water and rain water). This solubilization willrender the nutrients in the materials more accessible to the area inneed of fertilization.

In some embodiments, the methods described herein can be used to modifybiomass into materials for use as fertilizers. Such materials can becombined (e.g., blended) with seeds, nitrates, nitrites, nitrogen,phosphorus, potassium, calcium, lime, vitamins, minerals, pesticides,and any combinations thereof. Alternatively or in addition, suchmaterials can be combined with one or more microorganisms capable ofdegrading the materials and/or one or more enzymes capable of breakingdown the materials. These components can be provided together orseparately in liquid or dry forms. In some instances, these materialscan be associated with a structure or carrier such as netting, amembrane, a flotation device, a bag, a shell, or a biodegradablesubstance. Optionally, the structure or carrier itself can be made ofthe materials described herein. In some embodiments, these materials andcombinations of these materials can be mixed in a vessel (e.g., a bag orsolid container), e.g., to promote decomposition. Such mixtures can besupplied for use in a vessel (e.g., a bag or solid container).

In some embodiments, the methods described herein can be used togenerate materials that can be combined with plant seeds. For example,materials generated using a method described herein can be coated on thesurface of seeds, e.g., to protect seeds from rot, to protect seeds frommicroorganisms, and/or to fertilize seeds.

Chemical and Biological Applications

In some embodiments, the methods described herein can be used togenerate materials suitable for use as acids, bases, and/or buffers.Such materials can be used, e.g., to alter and/or buffer the pH of amaterial (e.g., a solid or liquid) in need of such treatment. Suchmaterials include solids and liquids not suitable for consumption and/orsolids and liquids intended for consumption (e.g., food products such asmeats, beverages, and dairy products).

In some embodiments, the methods described herein can be used togenerate materials suitable for use in maintaining or promoting thegrowth of microorganisms (e.g., bacteria, yeast, fungi, protists, e.g.,an algae, protozoa or a fungus-like protist, e.g., a slime mold), and/orplants and trees.

Lignin

In some embodiments, the methods described herein can also be used togenerate lignin, e.g., lignin residue.

Lignin is a phenolic polymer that is typically associated with cellulosein biomass, e.g., plants. In some instances the methods described hereinwill generate lignin that can be obtained (e.g., isolated or purified)from the biomass feedstock described herein. In some embodiments, thelignin obtained from any of the processes described herein can be, e.g.,used as a plasticizer, an antioxidant, in a composite (e.g., a fiberresin composite), as a filler, as a reinforcing material, and in any ofthe pharmaceutical compositions described herein.

In addition, as described above, lignin-containing residues from primaryand pretreatment processes has value as a high/medium energy fuel andcan be used to generate power and steam for use in plant processes.However, such lignin residues are a new type of solid fuel and there maybe little demand for it outside of the plant boundaries, and the costsof drying it for transportation may subtract from its potential value.In some cases, gasification of the lignin residues can be used toconvert it to a higher-value product with lower cost.

In some embodiments, lignin can be combined with one or more of theproducts and co-products described herein. For example, lignin can becombined with one or more herbicides and/or pesticides, e.g., togenerate a slow release system, e.g., where one or more herbicidesand/or pesticides are released over a period of time. Such slow releasesystems can be combined with the fertilizers described herein.Alternatively or in addition, lignin can be combined with charged (e.g.,positively or negatively charged) materials to generate a slow releaseair purification system. In some embodiments, lignin can be used, e.g.,alone or in combination with one or more of the products and co-productsdescribed herein, as a composite, e.g., for use as a plastic additiveand/or a resin.

An example of the structure of a lignin is shown below.

Other Products

Cell matter, furfural, and acetic acid have been identified as potentialco-products of biomass-to-fuel processing facilities. Interstitial cellmatter could be valuable, but might require significant purification.Markets for furfural and acetic acid are in place.

Bioconversion Products

As described above, the methods described herein can be used to processbiomass to obtain/produce, for example, foodstuffs (e.g., animal(including aquatic), human, and/or microbial foodstuffs), proteins, fatsand oils, carbohydrates and sugars, vitamins, minerals, ash,pharmaceuticals, nutriceuticals and nutraceuticals, pharmaceuticaldosage forms, hydrogels, absorbent materials, air purificationmaterials, food preservatives, herbicides and pesticides, fertilizers,acids, bases and buffers, and lignin. As shown in FIG. 43A, in general,these methods involve processing biomass, e.g., changing (e.g.,lowering) the recalcitrance level of the biomass, to obtain products,e.g., derived directly from the biomass and/or to produce productscomprising these materials.

Alternatively or in addition, the methods described herein can be usedto process a first material (e.g., biomass), e.g., to change (e.g.,lower) the recalcitrance level of the biomass, to produce a secondmaterial that can be used as a substrate for additional processes, e.g.,to generate materials and products present (e.g., substantially present)or abundant in the first material. In some embodiments, the additionalprocesses can include a bioconversion step as shown in FIG. 43B. In someembodiments, the bioconversion step can include the use ofmicroorganisms. Examples of methods including a bioconversion step aredescribed above, for example, in the use of the methods described hereinto produce energy products (e.g., ethanol), alcohols, and/or organicacids, all of which are not necessarily present (e.g., not substantiallypresent) or abundant in natural unprocessed biomass. Further examples ofsuch methods are described below.

Edible Products

In some embodiments, the methods described herein can be performed incombination with a bioconversion step (e.g., see FIG. 43B) to produce anedible product (e.g., an ingestible product such as a food product,e.g., an edible starch and/or protein) for use with animals or humans.One advantage of such methods over conventional agricultural foodproduction methods is that the methods described herein do not requirelarge areas of land and can be performed in environments that do notfavor conventional food production methods.

Malnutrition, particularly protein calorie malnutrition, is a increasingproblem around the world, especially in the developing world.Insufficient calories and protein contribute to increased infectiousdisease, stunt physical growth, and retard brain and mental development.These malnutrition problems are caused by increasing global populationscoupled with inadequate food supplies in developing countries and agingfood production methods. Without change in population growth, supplies,and food production methods, malnutrition will also become a seriousproblem within developed countries. One solution to these problems is toincrease food supply. This will be difficult under conventionalagricultural practice, however, due to limited availability of land foragriculture and the well-documented changing global climate. Inaddition, conventional agricultural practices are not favorable incertain environments, for example, environments that present excessiveheat or cold, limited oxygen, and/or limited sunlight. An alternativesolution is to modify the usage of currently available materials (e.g.,biomass) to create alternative food supplies, for example, to increasethe nutritional value or usability of already available materials.

The use of microbial proteins as a food for consumption by animals andhumans is known in the art and is monitored by The Food and AgricultureOrganization of the United Nations (FAO). The FAO in collaboration withthe World Health Organization (WHO) has published several publiclyavailable reports outlining guidelines and the standards required forfoods derived from biotechnology (see, e.g., Joint FAO/WHO ExpertConsultation on Foods Derived from Biotechnology, 1996; Steve Taylor,Joint FAO/WHO Expert Consultation on Foods Derived from Biotechnology,2001 (Biotech 01/03); David Ow, Joint FAO/WHO Expert Consultation onFoods Derived from Biotechnology, 2000 (Biotech 00/14)). Theseguidelines outline the safety issues to be considered when usingmicroorganisms to produce foods, types of organisms that are suitablefor such application, and the requirements of the proteins produced(see, e.g., Commission of Genetic Resources for Food and Agriculture,11^(th) Session, Rome Jun. 11-15, 2007, publication referenceCGRFA-11/07/Circ.3).

The use of microbes and microbial proteins as a food source is supportedby their known long-term use as foods. For example, the Indonesian plantTempeh is combined with the fungus (e.g., mold) Rhizopus oligosporus andconsumed. Algae are used as a source of food by shore side populationsof Lake Chad and Lake Texcoco in Mexico, and several thousand tons ofspirulina are now produced as a protein rich food source in Mexico. Inthe mid 1960s, a quarter of a million tons of food yeast were beingproduced and the Soviet Union planned an annual production of 900,000tons of food yeast by 1970 to compensate for agricultural proteindeficits (Bunker, “New Food,” 2nd Int. Congr. Food Sci. and Technol.,Warsaw. p. 48 (1966)). Due to marked improvements in crop production,increased communication between countries with food surpluses anddeficits, and the increasing cost of oil, microbial protein productiondid not develop as forecast. Nevertheless, protein derived from thefungus Fusarium venenatum is currently approved for consumption inEurope and is sold in the U.S. under the trademark Quorn® (for a reviewsee Wiebe, Mycologist, 18:17-20, 2004).

The use of microbial proteins as a food source for animals and humans isfurther supported by the observation that the chemical composition andlevels of microbial protein from bacteria, fungi (e.g., yeast and mold),and algae is comparable to that of soybean oilmeal. Furthermore, theamino acid composition and digestibility (including total energy(kcal/kg) based on data collected in pigs) of microbial proteins fromyeast, bacteria, fungi, and algae is also reported to be comparable tosoybean oilmeal (see, for example, Young et al., U.S. Pat. No.4,938,972).

In some embodiments, the food products described below can be producedusing a fed-batch fermentation process in which nutrients are added in acontrolled manner in accordance with the requirements of the culturesolution.

Proteins

Methods for obtaining microbial proteins using cellulosic materials aredescribed in the art (see, e.g., Ramasamy et al., J. Appl. Biotechnol.,46:117-124, 1979, Young et al., Biotechnol Lett., 14:863-868, 1992,Anupama and Ravindra, Brazilian Archives or Biology and Biotechnol.,44:79-88, 2001, U.S. Pat. Nos. 3,627,095, 4,379,844, 4,447,530,4,401,680, 4,526,721, 5,047,332, and 4,938,972).

In some embodiments, the methods described herein can be performed incombination with a bioconversion step (e.g., see FIG. 43B) to produceproteins. In some embodiments, the second material is used as asubstrate for microorganisms, which convert the organic matter presentin the second material into proteins, e.g., microbial proteins (e.g.,when combined with a nitrogen source). In some embodiments, the proteinscan be used as or in ingestible products (e.g., foods) for consumptionby animals and/or humans.

The term microbial proteins includes single cell proteins (SCP), a termcoined in the 1960s to embrace microbial biomass produced byfermentation in which the microbial cells are generally isolated fromthe substrate, and microbial biomass products (MBP), a material in whichthe substrate is not purified from the SCP.

Exemplary microbial proteins can be obtained from cells of bacteria,fungi (e.g., yeasts and moulds), and or algae. When cultured correctly,these cells can contain in excess of 40% protein on a dry weight basis.One advantage of using microbial proteins as a potential food source isthat microbial protein is a readily renewable and easily obtainableresource. For example, 1000 kg of yeast can produce 12000 kg of newcells containing 6000 kg of protein in 24 hours.

In some embodiments, microbial proteins can be produced using themethods described herein to process a first material (e.g., biomass)into a second material (e.g., a substrate) that is supplied to one ormore of a bacteria, fungus (e.g., yeasts and mould), and/or algae, e.g.,in the presence of nitrogen or a nitrogen source, in the presence orabsence of oxygen and at a temperature and pH, as required by theorganism or mixture of organisms to synthesize protein (e.g., at a levelabove the normal level of protein synthesis in the cell). In general,these methods include the use of any microorganism that synthesizesprotein in the presence of the materials generated using the methodsdescribed herein. Such organisms will typically be suitable or capableof being made suitable for consumption by animals and/or humans. In someembodiments, the microorganism can be non-pathogenic and/or an organismthat is generally recognized as safe (GRAS). Additional selectioncriteria to be considered when choosing a microorganism can include, forexample, consideration of whether the organism is capable of or can bemodified to produce large quantities of proteins (e.g., edible proteinsor proteins that can be rendered edible); whether isolated cultures ofthe organism are commercially available and/or whether the organism canbe efficiently isolated; whether the microorganism can be readilymaintained in culture; whether the microorganism is genetically stable;and whether the organism can efficiently utilize the substratesgenerated using the methods described herein (e.g., whether themicroorganism can be cultured on the supplied substrate).

In some embodiments, the microorganisms can be modified (e.g.,engineered) to express one or more recombinant proteins, for example,proteins that are not normally encoded by the microorganisms. Forexample, these proteins can be proteins known to be of a highnutritional value for humans and/or animals (e.g., as determined byassessing the biological value (BV) of a protein (e.g., the proportionof the absorbed nitrogen retained) and/or net protein utilization (NPU)of a protein (e.g., the proportion of ingested protein retained). Inexperimental animals NPU can be directly estimated by carcass analysisand values are therefore likely to be more accurate than when BV and NPUare derived from N balance data, as it is done in human studies. Theinaccuracies inherent in N balance studies are known, no matter howcarefully conducted. NPU and BV thus measure the same parameter (Nretained, except that BV is calculated from N absorbed and NPU from Ningested (for a review see, e.g., Bender, Relation Between ProteinEfficiency and Net Protein Utiliization, Measurement of ProteinUtilization, 10: 135-143, 1956). In some embodiments, proteins of highnutritional value can have a high BV at an intake level (mg/kg) requiredto obtain the recommended daily protein requirement of the animal and/orhuman and can contain suitable levels of all essential amino acids (EAA)required for protein generation in the animal or human (EAAs includee.g., phenylalanine (FAO recommended daily intake is 2.2 g); methionine(FAO recommended daily intake is 2.2 g); leucine (FAO recommended dailyintake is 2.2 g); valine (FAO recommended daily intake is 1.6 g); lysine(FAO recommended daily intake is 1.6 g); isoleucine (FAO recommendeddaily intake is 1.4 g); threonine (FAO recommended daily intake is 1.0g); and tryptophan (FAO recommended daily intake is 0.5 g)). In someembodiments, the proteins of high nutritional value can be syntheticproteins, e.g., designed to have high BV at intake levels required toobtain the recommended daily protein requirement of the animal and/orhuman and can contain suitable levels of all EAAs required for proteingeneration in the animal or human. In some embodiments, proteins of highnutritional value can be labeled (e.g., tagged), e.g., to facilitateidentification and/or purification of the protein. Such proteins arealso referred to herein as microbial proteins.

Exemplary fungi that can be used in the methods described hereininclude, but are not limited to, Aspergillus niger, A. funigatus, A.terreus, Cochliobolus specifer, Myrothecium verrucaria, Rhizoctoniasolani, Spicaria fusispora, Penicillium sp., Gliocladium sp., Fusariumsp., Trichosporon cutaneum, Neurospora sitophila, Chaetomiiumcellulolyticum, Fusarium venenatum (formally F. graminearum) strain A3/5 (e.g., ATCC 20334. Suitable culture conditions for this organism aredisclosed in U.S. Plant Patent No. 4347 and European Patent No.123,434). F. solani, F. oxysporium, and Paecilomyces variotii, mycelium,Rhizopus oligosporus, Candida utilis, and Saccharomyces cerevisiae.Exemplary algae that can be used in the methods described hereininclude, but are not limited to, Spirulina sp., Scenedesmus acutus,Spirulina maxima, and Cosmarium turpinii. Exemplary bacteria that can beused in the methods described herein include, but are not limited to,Rhodospirillum sp., and Rhodopseudomonas sp., Corynebacteriumglutamicum, Escherichia coli, Alcaligenes faecalis, Thermomonosporafusca (Actinomycetaceae) and Pseudomonas JM127.

In some embodiments, microbial proteins can be fed to animals and/orhumans as SCP, e.g., without isolation from the microorganism or mixtureof microorganisms. In such cases, SCP containing cells can beconcentrated using, for example, filtration, precipitation, coagulation,centrifugation, and the use of semi-permeable membranes. SCP containingcells can also be dried, e.g., to about 10% moisture and/or condensedand acidified to limit spoilage. In some embodiments, SCP can be fed toanimals and/or humans shortly (e.g., within 12 hours, 24 hours, 48hours) after production without further treatment of the SCP. In someembodiments, SCP can be consumed in the absence of further food sources(see the FAO publication for the recommended daily intake of SCP byanimals and humans). Alternatively or in addition, SCP can be combined,e.g., mixed with other food sources prior to or at the same time asconsumption by an animal and/or human. SCP can be combined with dryand/or wet food sources to create SCP mixtures. In some embodiments,SCP-containing mixtures can be processed, e.g., as described byTannenbaum (U.S. Pat. No. 3,925,562). For example, SCP microorganismscan be combined with a protein complement (e.g., vegetable protein) andtexturized into a paste suitable for use as a food additive. Suchprocesses can be used to add desirable texture properties to SCP.

In some embodiments, the protein utilization and nitrogen digestibilityof SCP proteinaceous material can be increased by homogenizing the cells(see, for example, Yang et al., J. Food Sci., 42:1247-1250, 2006). Thus,in some embodiments, microbial proteins can be extracted or isolatedfrom the microorganism or mixture of microorganisms prior to consumptionby animals and/or humans. For example, microbial proteins can beextracted by chemically, enzymatically, and/or mechanically disruptingthe microbial cell wall and/or membranes, e.g., to release theintracellular contents of the cells. Microbial proteins can then beisolated or purified from contaminating materials using proteinisolation techniques known in the art. In some embodiments, microbialproteins can be isolated or purified by way of a detectable tag fused tothe protein.

In some embodiments, microbial proteins can be modified, e.g.,glycosylated and/or folded prior to use, e.g., to make them more or lessantigenic.

In some embodiments, microbial proteins can be isolated and hydrolyzedto single amino acids, peptides, and/or polypeptide, e.g., prior toconsumption by animals and/or humans. Methods for protein hydrolysis areknown in the art.

In some embodiments, microbial proteins can be purified (to at least50%,e.g., to 60%, 70%, 80%, 90%, 95%, 99% or 100% weight/weight,weight/volume, or volume/volume) and optionally concentrated. Thestructure of the proteins can then be modified to resemble the fibrousstructure of animal muscle protein before the product is flavored usingmeat flavors and fats. In some embodiments, microbial proteins can beused as the primary protein source in a meat analogue. Alternatively,microbial proteins can be used to supplement currently commerciallyavailable meat analogues, for example, those sold under the tradenameQuorn® and soy protein based products.

Fats, Oils, Lipids and Hydrocarbons

In some embodiments, the methods described herein can be performed incombination with a bioconversion step (e.g., see FIG. 43B) to generatefats and/or oils.

The market place for fats and oils is large and extremely diversified,ranging from bulk commodities used for food and technical purposes tomore specialized oils. The use of microbial fats and oils is known inthe art (for a review on this topic see, e.g., Pryde, New Sources ofFats and Oils, Amer Oil Chemists Society, (American Oil Chemist Society(AOCS), 1981).

In some embodiments, the fats and/or oils generated using the methodsdescribed herein can be used, for example, as substitutes for animal andplant based fats and oils, in the production of energy products,flammables (solid and/or liquid), in food preparation and cooking, asflavor enhancers (e.g., for food products), as or in animal feed, as orin food supplements, as or in pharmaceuticals, as or in nutriceuticals,as or in cosmetics, and as or in post operative nutritive therapy.

In some embodiments, microbial fats and/or oils can be produced usingthe methods described herein to process a first material (e.g., biomass)into a second material (e.g., a substrate) that is supplied to one ormore of a bacteria, fungi (e.g., yeasts and moulds), and/or algae, inthe presence or absence of oxygen and at a temperature and pH, asrequired by the organism or mixture of organisms to synthesize fatsand/or oils (e.g., at a level above the normal level of fat and/or oilsynthesis in the cell). In general, these methods include the use of anymicroorganism that synthesizes fats and/or oils in the presence of thematerials generated using the methods described herein. In someembodiments, the microorganism can be non-pathogenic and/or an organismthat is generally recognized as safe (GRAS). Additional selectioncriteria to be considered when choosing a microorganism include, forexample, consideration of whether the organism is capable of producingor can be modified to produce large quantities of fats and oils; whetherisolated cultures of the organism are commercially available and/orwhether the organism can be efficiently isolated; whether themicroorganism can be readily maintained in culture; whether themicroorganism is genetically stable; and whether the organism canefficiently utilize the substrates generated using the methods describedherein (e.g., whether the microorganism can be cultured on the suppliedsubstrate).

In some embodiments, microorganisms that can be used in the methodsdescribed herein, e.g., to generate or produce microbial fats and/oroils include, for example, bacteria (e.g., mycobacteria, corynebacteria,and norcardia), algae (e.g., Chlorophyta (Cladophora rupestris,Blidingia minima, Enteromorpha intestinalis), Phaeophyta (Agarumcribrosum, Ascophyllum nodosum, and Laminaria digitata), and Rhodophyta(Polysiphonia lanosa, palmaria palmate, Halosaccion ramentaceum, andPorphyra leucosticte)), seaweeds and seagrasses, yeast (e.g., Candida107, Crytococcus terricolus, Hansenula saturnus, Lipomyces lipofera, L.starkeyi, Rhodotorula gracilis, R. toruloides, and Candida curvata), andmolds (e.g., Aspergillus nidulans, A. terreus, Fusarium monoiliforme,Mucor circinelloides, Penicillium spinulosum, Rhizopus sp.),

In some embodiments, microbial fats and/or oils generated using themethods disclosed herein can be separated, e.g., isolated from themicrobial cells prior to use. Alternatively or in addition, themicrobial fats and oils generated using the methods disclosed herein canbe used without being separated from the microbial cells.

Some microorganisms can be used to produce hydrocarbons. For example, asdiscussed in the Background section of U.S. 2008/0293060, the disclosureof which is incorporated herein by reference, numerous organisms, suchas bacteria, algae and plants, can synthesize hydrocarbons, e.g.n-alkanes of various carbon chain lengths, as previously described(Dennis, M. W. & Kolattukudy, P. E. (1991) Archives of biochemistry andbiophysics 287, 268-275; Kunst, L. & Samuels, A. L. (2003) Progress inlipid research 42, 51-80; Tillman, J. A., Seybold, S. J., Jurenka, R.A., & Blomquist, G. J. (1999) Insect biochemistry and molecular biology29, 481-514; Tornabene, T. G. (1982) Experientia 38.1-4, each of whichis incorporated by reference).

Exemplary species that synthesize hydrocarbons are listed in Table A andTable B below.

TABLE A Hydrocarbon producing prokaryotes Strain ATCC # or ReferenceMicrococcus luteus ATCC 272 Micrococcus luteus ATCC 381 Micrococcusluteus ATCC 398 Micrococcus sp. ATCC 401 Micrococcus roseus ATCC 412Micrococcus roseus ATCC 416 Micrococcus roseus ATCC 516 Micrococcus sp.ATCC 533 Micrococcus luteus ATCC 540 Micrococcus luteus ATCC 4698Micrococcus luteus ATCC 7468 Micrococcus luteus ATCC 27141Jeotgalicoccus sp. ATCC 8456 Stenotrophomonas maltophilia ATCC 17674Stenotrophomonas maltophilia ATCC 17679 Stenotrophomonas maltophiliaATCC 17445 Stenotrophomonas maltophilia ATCC 17666 Desulfovibriodesulfuricans ATCC 29577 Vibrio furnissii M1 Park, 2005, J. Bact., vol.187, 1426-1429 Clostridium pasteurianum Bagaeva and Zinurova, 2004,Biochem (Moscow), vol. 69, 427-428 Anacystis (Synechococcus) Winters etal., 1969, Science, vol. 163, nidulans 467-468 Nostoc muscorum Winterset al., 1969, Science, vol. 163, 467-468 Cocochloris elabens Winters etal., 1969, Science, vol. 163, 467-468 Chromatium sp. Jones and Young,1970, Arch. Microbiol., vol. 70, 82-88

TABLE B Hydrocarbon producing eukaryotes Organism ATCC # or ReferenceCladosporium resinae ATCC 22711 Saccharomycodes ludwigii ATCC 11311Saccharomyces cerevisiae Baraud et al., 1967, Compt. Rend. Acad. Aci.Paris, vol. 265, 83-85 Botyrococcus braunii Dennis and Kolattukudy,1992, PNAS, vol. 89, 5306-5310 Musca domestica Reed et al., 1994, PNAS,vol. 91, 10000- 10004 Arabidopsis thaliana Aarts et al., 1995, PlantCell, vol. 7, 2115-2127 Pisum sativum Schneider and Kolattukudy, 2000,Arch. Biochem. Biophys., vol. 377, 341-349 Podiceps nigricollisCheesborough and Kolattukudy, 1988, J. Biol. Chem., vol 263, 2738-2743

Carbohydrates, Sugars, Biopolymers, and Polymer Precursors

A large variety of biopolymers, for example, such as polysaccharides,polyesters, and polyamides, are naturally produced by microorganisms(for a review see Microbial Production of Biopolymers and PolymerPrecursors, Rehm, ed, (Caister Academic Press, 2009)). These biopolymersrange from viscous solutions to plastics and their physical propertiesare dependent on the composition and molecular weight of the polymer.

In some embodiments, the methods described herein can be performed incombination with a bioconversion step (e.g., see FIG. 43B) to generatecarbohydrates, sugars, biopolymers, and polymer precursors. In someembodiments, the methods described herein can be used to process a firstmaterial (e.g., biomass) to generate a second material that can be usedas a substrate for microorganisms (e.g., bacteria, fungi (e.g., yeastsand moulds), and/or algae) capable of generating, for example, xanthan,alginate, cellulose, cyanophycin, poly(gamma-glutamic acid), levan,hyaluronic acid, organic acids, oligosaccharides and polysaccharides,and polyhydroxyalkanoates. Uses of these carbohydrates, sugars,biopolymers, and polymer precursors include, for example, as foodadditives, in cosmetics, in plastic manufacturing, in fabricmanufacturing, and in pharmaceutical and nutraceuticals.

In general, these methods include the use of any microorganism thatsynthesizes one or more of carbohydrates, sugars, biopolymers, and/orpolymer precursors in the presence of the materials generated using themethods described herein. In some embodiments, these methods include theuse of any microorganism that synthesizes one or more of xanthan,alginate, cellulose, cyanophycin, poly(gamma-glutamic acid), levan,hyaluronic acid, organic acids, oligosaccharides and polysaccharides,and polyhydroxyalkanoates in the presence of the materials generatedusing the methods described herein. In some embodiments, suitableorganisms will be suitable or capable of being made suitable forconsumption by animals and/or humans or will be generally recognized assafe (GRAS).

Additional selection criteria to be considered when choosing amicroorganism include, for example, consideration of whether theorganism is capable or can be modified to produce large quantities ofone or more of carbohydrates, sugars, biopolymers, and/or polymerprecursors (e.g., xanthan, alginate, cellulose, cyanophycin,poly(gamma-glutamic acid), levan, hyaluronic acid, organic acids,oligosaccharides and polysaccharides, and polyhydroxyalkanoates);whether isolated cultures of the organism are commercially availableand/or whether the organism can be efficiently isolated; whether themicroorganism can be readily maintained in culture; whether themicroorganism is genetically stable; and whether the organism canefficiently utilize the substrates generated using the methods describedherein (e.g., whether the microorganism can be cultured on the suppliedsubstrate).

Vitamins

In some embodiments, the methods described herein can be performed incombination with a bioconversion step (e.g., see FIG. 43B) to generatevitamins, for example, including, but not limited to, vitamin Riboflavin(vitamin B2), vitamin B12, and vitamin C.

In some embodiments, the substrate is used by the microorganism AshbyagossifYii and the vitamin generated is Riboflavin (vitamin B2).

In some embodiments, the substrate is used by the microorganismsBacillus megatherium, Pseudomonas denitrificans, and/or species of thegenus Propionibacterium and the vitamin generated is vitamin B12.

In some embodiments, the substrate is used by the microorganismSaccharomyces sp. and the vitamin generated is vitamin C.

In some embodiments, vitamin products can be produced using a fed-batchfermentation process in which nutrients are added in a controlled mannerin accordance with the requirements of the culture solution.

Mushrooms

In some embodiments, the methods described herein can be used to processa first material (e.g., biomass), e.g., to change (e.g., lower) therecalcitrance level of the biomass, to produce a second material thatcan be used as a substrate for the cultivation or growth mushrooms.These mushrooms can be used as a higher quality food source than thefirst material (e.g., biomass) and the second material that can beingested by animals and/or humans as a food.

Mushrooms are fungi that grow above ground on a suitable food source. Asused herein, the term mushroom refers to edible mushrooms including, butnot limited to, fungi with a stem (stipe), a cap (pileus), and gills(lamellae) on the underside of the cap and fungi without stems, thefleshy fruiting bodies of some Ascomycota, the woody or leatheryfruiting bodies of some Basidiomycota, and spores of edible mushrooms.In some embodiments, the term mushroom includes fungi edible to animals.

In some embodiments, mushrooms useful in the present disclosure include,but are not limited to, for example, mushrooms, mushroom mycelia, andmushroom spores of the mushrooms Pleurotus sajor-caju, Basidiomycota,Agaricomycetes, Vilvariella volvacea (the padi mushroom), Pleurotusostreatus (the oyster mushroom), Agaricus bisporus, Flammulinavelutipes, Pleurotus eryngii, Ganoderma mushrooms and Cordyceps.

Methods for cultivating mushrooms are known in the art (see, e.g., U.S.Pat. No. 6,737,065). Following cultivation, mushrooms can be harvestedand stored for later use or can be used immediately. Mushrooms haverelatively low protein content (e.g., 2-5%) on a fresh weight basis,however, the protein content of mushrooms can be increased by drying themushrooms (e.g., 30-50% on dry weight basis). In some embodiments,therefore, mushrooms generated using the methods described herein can bedried (e.g., freeze dried) or dehydrated prior to use, e.g., ingestion.In some embodiments, mushrooms can be mixed with a protein complementand binding agent and can be textured.

Hydroponics

In some embodiments, the methods described herein can be used to processa first material (e.g., biomass), e.g., to change (e.g., lower) therecalcitrance level of the biomass, to produce a second material thatcan be used in hydroponics. Hydroponics is a method of growing plantsusing mineral nutrient solutions, without soil. Plants may be grown withtheir roots in the mineral nutrient solution only (solution culture) orin an inert medium (medium culture), such as perlite, gravel, or mineralwool. The three main types of solution culture are static solutionculture, continuous flow solution culture and aeroponics. Materialsformed using the processes disclosed herein can be used alone orcombined with macronutrients, e.g., potassium nitrate, calcium nitrate,potassium phosphate, and magnesium sulfate, to form a hydroponicsolution. Various micronutrients may also be included to supplyessential elements, e.g., Fe (iron), Mn (manganese), Cu (copper), Zn(zinc), B (boron), Cl (chlorine), and Ni (nickel). Chelating agents maybe added to enhance the solubility of iron. Different hydroponicsolutions may be utilized throughout the plant life cycle to enhancegrowing conditions.

Aquaculture

In some embodiments, the methods described herein can be used to processa first material (e.g., biomass), e.g., to change (e.g., lower) therecalcitrance level of the biomass, to produce a second material thatcan be used in aquaculture. For example, the second material can be usedto feed or otherwise maintain aquatic species. Aquaculture is thefarming of freshwater and saltwater organisms including mollusks,crustaceans and aquatic plants. Unlike fishing, aquaculture, also knownas aquafarming, implies the cultivation of aquatic populations undercontrolled conditions. Mariculture refers to aquaculture practiced inmarine environments. Particular kinds of aquaculture include algaculture(the production of kelp/seaweed and other algae), fish farming, shrimpfarming, oyster farming, and the growing of cultured pearls. Aquaponicsintegrates fish farming and plant farming using the symbioticcultivation of plants and aquatic animals in a recirculatingenvironment.

Production of Edible Fusarium venenatum

In some embodiments, the methods described herein can be used to processa first material (e.g., biomass), e.g., to change (e.g., lower) therecalcitrance level of the biomass, to produce a second material thatcan be used as a substrate that can be used as a substrate for thegeneration of edible Fusarium venenatum (e.g., which is marketed underthe trade name Quorn®). Methods for producing Quorn® are described, forexample, in U.S. Pat. Nos. 5,935,841, 6,270,816, 5,980,958, and3,809,614, and are reviewed in Weibe (Weibe, Mycologist, 18:17-20,2004). Current Quorn® production methods use glucose as the primarycarbon source. Substituting glucose with the substrate described hereinwould reduce the cost associated with Quorn® production as thesubstrates provided herein provide a cheaper carbon source than glucose.

Alcoholic Beverages

In some embodiments, the methods described herein can be used to processa first material (e.g., biomass), e.g., to change (e.g., lower) therecalcitrance level of the biomass, to produce a second material thatcan be used as a substrate for the generation of alcohol that issuitable for consumption by humans. Such alcohols can be used as or inthe production of alcoholic beverages. For example, alcohols producedusing the methods described herein can be used as or in the productionof beers, wines, spirits, and/or alcopops.

Health Products

In some embodiments, the methods described herein can be used to processa first material (e.g., biomass), e.g., to change (e.g., lower) therecalcitrance level of the biomass, to produce a second material thatcan be used as a substrate as or in the generation of health productsfor animal or human use. Such health products can include, for example,pharmaceuticals, nutriceuticals, cosmetics, cosmeceuticals, and beautyproducts (e.g., creams and lotions (e.g., for use on skin and/or hair)).In some embodiments, these health products can include, for example,functional foods that do not necessarily provide any nutritional value,but that increase motility of the gastrointestinal tract, or that can beused to reduce cholesterol levels (e.g., high fiber products includingsoluble and/or insoluble fiber and soluble and/or insoluble fibercontaining products).

Amino Acids and Amino Acid Derivatives

Biotechnological processes have been used in the industrial productionof amino acids for 50 years (for a recent review see Leuchtenberger etal., Appl. Microbiol. Biotechnol., 69:1-8, 2005). Major products includeflavor enhancers and animal feed products such as _(L)-lysine,_(L)-threonine, and _(L)-tryptophan, which are commonly, produced usinghigh-performance strains of Corynebacterium glutamicum (see Kinoshita etal., Gen. Appl. Microbiol., 3:193-205, 1957, and Kalinowshki et al., J.Biotechnol., 104:5-25, 2003) and Escherichia coli and substrates such asmolasses, sucrose, or glucose (Leuchtenberer, supra).

In some embodiments, the methods described herein can be used to processa first material (e.g., biomass), e.g., to change (e.g., lower) therecalcitrant level of the biomass, to produce a second material that canbe used as a substrate for microorganisms (e.g., bacteria, fungi (e.g.,yeasts and moulds), and/or algae) capable of generating amino acidsand/or amino acid derivatives (e.g., when combined with a nitrogensource). These amino acids and derivatives can be used, for example, asflavor enhancers (e.g., for food products), in animal feed, as foodsupplements, and in the production of pharmaceuticals, nutriceuticals,cosmetics, and in post-operative nutritive therapy.

In some embodiments, amino acids and amino acid derivatives that can beexpressed using the methods described herein include, but are notlimited to, for example, _(L)-amino acids and _(D)-amino acids such as_(L)-glutamic acid (monosodium glutamate (MSG)), _(L)-apartic acid,_(L)-phenylalanine, _(L)-lysine, _(L)-threonine, _(L)-tryptophan,_(L)-valine, _(L)-leucine, _(L)-isoleucine, _(L)-methionine,_(L)-histidine, and _(L)-phenylalanine, _(L)-lysine, _(DL)-methionine,and _(L)-tryptophan.

For example, the aromatic amino acids tryptophan, phenylalanine, andtyrosine are biosynthesized from glucose through the shikimic acidpathway (shown in FIG. 47).

The shikimic acid pathway converts simple carbohydrate precursorsderived from glycolysis and the pentose phosphate pathway to thearomatic amino acids. One of the pathway intermediates is shikimic acid,which lends its name to this entire sequence of reactions. The shikimicacid pathway is present in plants, fungi, and bacteria but is not foundin animals. Animals have no way to synthesize the three aromatic aminoacids-phenylalanine, tyrosine, and tryptophan-, which are thereforeessential nutrients in animal diets.

In some embodiments, these amino acids can be modified to produce aminoacid derivatives. Amino acid derivatives include, but certainly are notlimited to the following groups.

In some embodiments, microorganisms (e.g., bacteria, fungi (e.g., yeastsand molds), and/or algae) suitable for use in the generation of aminoacids can be, but are not limited to, non-pathogenic organisms and/ororganisms that are GRAS. Additional selection criteria to be consideredwhen choosing a microorganism include, for example, consideration ofwhether the organism is capable of producing or can be modified toproduce large quantities of a single product; whether isolated culturesof the organism are commercially available and/or whether the organismcan be efficiently isolated; whether the microorganism can be readilymaintained in culture; whether the microorganism is genetically stable;and whether the microorganism can be cultured on the supplied substrate.Alternatively or in addition, the microorganism can be a wild type(e.g., unmodified) or genetically modified microorganism (e.g., amutant), for example, a microorganism that has or can be modified toover-express one or more selected amino acids and/or amino acidderivatives. Exemplary microorganisms include, but are not limited to,lactic acid bacteria (LAB), E. coli, Bacillus subtilis, andCorynebacterium glutamicum (e.g., ATCC 13032).

In some embodiments, amino acids and amino acid derivatives can beexpressed using a fed-batch fermentation process in which nutrients areadded in a controlled manner in accordance with the requirements of theculture solution. In some embodiments, the methods and/or materialsdescribed herein can be incorporated into the processes currently usedby Ajinomoto (Japan), ADM (U.S.A.), Cheil-Jedang (South Korea), GlobalBioChem (China), and BASF and Degussa (Germany) in the generation ofamino acids and amino acid derivatives.

Antibiotics

In some embodiments, the methods described herein can be used to processa first material (e.g., biomass), e.g., to change (e.g., lower) therecalcitrance level of the biomass, to produce a second material thatcan be used as a substrate by microorganisms (e.g., bacteria, fungi(e.g., yeasts and moulds), and/or algae) capable of generatingantibiotics, for example, including, but not limited to, tetracycline,streptomycin, cyclohexamide, Neomycin, cycloserine, erythromycin,kanamycin, lincomycin, nystatin, polymyxin B, bacitracin, daptomycin,vancomycin, and the ansamycins or the natural products presented inFIGS. 48A through 48F.

In some embodiments, the substrate is used by the microorganismStreptomyces remosus and the antibiotic generated is tetracycline.

In some embodiments, the substrate is used by the microorganismStreptomyces griseus and the antibiotic generated is streptomycin and orcyclohexamide. The biosynthesis of streptomycin is illustrated in FIG.49 starting from D-glucose.

In some embodiments, the substrate is used by the microorganismStreptomyces frodiae and the antibiotic generated is neomycin.

In some embodiments, the substrate is used by the microorganismStreptomyces orchidaceus and the antibiotic generated is cycloserine.

In some embodiments, the substrate is used by the microorganismStreptomyces erythreus and the antibiotic generated is erythromycin.

In some embodiments, the substrate is used by the microorganismStreptomyces kanamyceticus and the antibiotic generated is kanamycin.

In some embodiments, the substrate is used by the microorganismStreptomyces lincolnensis and the antibiotic generated is lincomycin.

In some embodiments, the substrate is used by the microorganismStreptomyces noursei and the antibiotic generated is nystatin.

In some embodiments, the substrate is used by the microorganism Bacilluspolymyxa and the antibiotic generated is polymyxin B.

In some embodiments, the substrate is used by the microorganism Bacilluslicheniformis and the antibiotic generated is bacitracin.

In some embodiments, the substrate is used by the microorganismStreptomyces roseosporus and the antibiotic generated is daptomycin.

In some embodiments, the substrate is used by the microorganismAmycolatopsis orientalis and the antibiotic generated is vancomycin. Thebiosynthesis of vancomycin starting from a glucose derivative is shownin FIG. 50.

In some embodiments, the substrate is used by the two Streptomyceshygroscopicus strains and the antibiotics generated belong to theansamycin family. The biosynthesis of the ansamycins starting from aglucose derivative is shown in FIG. 51.

Carotenoids

In some embodiments, the methods described herein can be used to processa first material (e.g., biomass) to generate a second material that canbe used as a substrate by microorganisms (e.g., bacteria, yeast, fungi,mould, and or algae) capable of generating carotenoids, including, forexample, β-carotene, lycopene, and astaxanthin. Carotenoids arewater-soluble natural pigments of 30-50 carbon atoms. The industrial useof carotenoids involves their application in nutrient supplementation,for pharmaceutical purposes, as food colorants, and in animal feeds.

Representative Carotenoids in Industry

In some embodiments, antibiotic products can be produced using afed-batch fermentation process in which nutrients are added in acontrolled manner in accordance with the requirements of the culturesolution.

Vaccines

In some embodiments, vaccines are immunostimulatory molecules (e.g.,small molecules, peptides, and/or antigenic molecules). In someembodiments, the methods described herein can be used to process a firstmaterial (e.g., biomass), e.g., to change (e.g., lower) the recalcitrantlevel of the biomass, to produce a second material that can be used as asubstrate by microorganisms (e.g., bacteria, fungi (e.g., yeasts andmoulds), and/or algae) capable of generating vaccines, including, forexample, flu vaccine (e.g., a universal flu vaccine, for example, theVaxInnate M2e universal influenza vaccine).

In some embodiments, vaccine products can be produced using a fed-batchfermentation process in which nutrients are added in a controlled mannerin accordance with the requirements of the culture solution.

Specialty Chemicals

In some embodiments, the methods described herein can be used to processa first material (e.g., biomass), e.g., to change (e.g., lower) therecalcitrant level of the biomass, to produce a second material that canbe used as a substrate by microorganisms (e.g., bacteria, fungi (e.g.,yeasts and moulds), and/or algae) capable of generating specialtychemicals, for example, thickeners, xanthan (E 415), acidity regulators,citric acid (E 330), natamycin (E 235), nisin (E 234), and lysozyme (E1105). In some embodiments, the methods described herein can be used toproduce fine chemicals, e.g., flavorings and aromatics.

In some embodiments, chemical products can be produced using a fed-batchfermentation process in which nutrients are added in a controlled mannerin accordance with the requirements of the culture solution.

Alcohols

In some embodiments, the methods described herein can be used to processa first material (e.g., biomass), e.g., to change (e.g., lower) therecalcitrant level of the biomass, to produce a second material that canbe used as a substrate by microorganisms (e.g., bacteria, fungi (e.g.,yeasts and moulds), and/or algae) capable of generating alcohols inaddition to the energy products (e.g., ethanol) disclosed above, forexample, including but not limited to acetone and butanol. In someembodiments, the substrate is used by the microorganism Clostridiumacetobutylicum and the alcohol generated is acetone. In someembodiments, the substrate is used by the microorganism Clostridiumacetobutylicum mutant IFP 904 (ATCC 39058) and the alcohols produced areacetone and butanol.

In some embodiments, alcohol products described herein can be producedusing a fed-batch fermentation process in which nutrients are added in acontrolled manner in accordance with the requirements of the culturesolution.

Acids and Bases

In some embodiments, the methods described herein can be used to processa first material (e.g., biomass), e.g., to change (e.g., lower) therecalcitrant level of the biomass, to produce a second material that canbe used as a substrate by microorganisms (e.g., bacteria, fungi (e.g.,yeasts and moulds), and/or algae) capable of generating acids and bases.In some embodiments, the substrate is used by the microorganismsAcetobacter and/or Gluconobacter and the acid generated is acetic acid(e.g., for use in the production of vinegar).

In some embodiments, acid and base products can be produced using afed-batch fermentation process in which nutrients are added in acontrolled manner in accordance with the requirements of the culturesolution.

Enzymes

In some embodiments, the methods described herein can be used to processa first material (e.g., biomass), e.g., to change (e.g., lower) therecalcitrant level of the biomass, to produce a second material that canbe used as a substrate by microorganisms (e.g., bacteria, fungi (e.g.,yeasts and moulds), and/or algae) capable of generating enzymes.

Exemplary enzymes that can be produced using the methods describedherein include, but are not limited to, e.g., rennet, glucoamylase,polygalacturonase, cellulase, alpha-amylase, protease, betaglucanase,pullulanase, amyloglucosidase, phospholipase, xylanase, mono glucoseoxidase, novo lipase, ultra lipase, lipase, maltogenic amylase,alpha-acetodecarboxylase, tender protease, pectinesterase, carbohydrase,cellobiose oxidase, lipase, pectin lyase, mono xylanase, transferase,wheat xylanase, phytase, subtillisin, It-l alpha-amylase, pectate,mannanase, trypsin, and laccase. The uses of such enzymes (e.g., aloneor in combinations of one or more of the enzymes) in, for example, thejuice industry, the brewing industry, the starch industry, the bakingindustry, the oils and fats industry, the meat industry, the dairyindustry, the alcohol industry, the animal feed industry, the detergentindustry, the textile industry, and the personal care industry are knownin the art.

In some embodiments, enzyme products can be produced using a fed-batchfermentation process in which nutrients are added in a controlled mannerin accordance with the requirements of the culture solution.

Growth Factors

In some embodiments, the methods described herein can be used to processa first material (e.g., biomass), e.g., to change (e.g., lower) therecalcitrant level of the biomass, to produce a second material that canbe used as a substrate by microorganisms (e.g., bacteria, fungi (e.g.,yeasts and moulds), and/or algae) capable of generating growth factors.

Exemplary growth factors that can be produced using the methodsdescribed herein include, but are not limited to, insulin-like-growthfactor, keratinocyte growth factor (KGF)-1 and -2, epidermal growthfactor, fibroblast growth factor, granulocyte-macrophagecolony-stimulating factor, human growth hormone, interleukin-1,platelet-derived growth factor, and transforming growth factor-β.

In some embodiments, growth factor products can be produced using afed-batch fermentation process in which nutrients are added in acontrolled manner in accordance with the requirements of the culturesolution.

Plastics

In some embodiments, the methods described herein can be used to processa first material (e.g., biomass), e.g., to change (e.g., lower) therecalcitrant level of the biomass, to produce a second material that canbe used as a substrate by microorganisms (e.g., bacteria, fungi (e.g.,yeasts and moulds), and/or algae) capable of generating plastics orplastic precursors. In some embodiments, the substrate is used by themicroorganism Alcaligenes eutrophas and the molecules generated arePoly-B-hydroxybutyrate and Poly-B-hydroxyvalerate.

In some embodiments, plastic products can be produced using a fed-batchfermentation process in which nutrients are added in a controlled mannerin accordance with the requirements of the culture solution.

Fertilizers

In some embodiments, the methods described herein can be used to processa first material (e.g., biomass), e.g., to change (e.g., lower) therecalcitrant level of the biomass, to produce a second material that canbe used as a substrate by microorganisms (e.g., bacteria, fungi (e.g.,yeasts and moulds), and/or algae) capable of generating materials thatcan be used as or in fertilizers (e.g., proteins, fats and oils,carbohydrates, and/or minerals). In some embodiments, fertilizersgenerated using the methods described herein can be protein-based orprotein-rich fertilizers (see Paungfoo-lonhienne et al., PNAS,104:4524-4529, 2008, for a review of protein-based fertilizers).

Culture Methods

As detailed above, the methods described herein can be used to process afirst material (e.g., biomass), e.g., to change (e.g., lower) therecalcitrant level of the biomass, to produce a second material that canbe used as a substrate by microorganisms (e.g., bacteria, fungi (e.g.,yeasts and moulds), and/or algae) to generate materials and products notnecessarily present (e.g., not substantially present) or abundant in thefirst material. The choice of microorganisms will depend on the productto be produced.

Microorganism Selection

Several additional factors can also be considered when selectingsuitable microorganisms for use in the methods described herein. Forexample, if the microorganisms are to be used to generate a healthproduct for use with animals or humans, or if the microorganisms are tobe used as or in the production of a food, the microorganisms selectedwill typically be non-pathogenic and/or generally recognized as safe(GRAS). In addition, the microorganisms selected should be capable ofproducing large quantities of the desired product or should be able tobe modified to produce large quantities of the desired product. In someembodiments, the microorganisms can also be commercially availableand/or efficiently isolated, readily maintainable in culture,genetically stable and/or well characterized. Selected microorganismscan be wild type (e.g., unmodified) or genetically modifiedmicroorganisms (e.g., mutated organisms). In some embodiments, agenetically modified microorganism can be adapted to increase itsproduction of the desired product and/or to increase the microorganismstolerance to one or more environmental and/or experimental factors, forexample, the microorganism can be modified (e.g., engineered) totolerate temperature, pH, acids, bases, nitrogen, and oxygen levelsbeyond a range normally tolerated by the microorganism. Alternatively orin addition, the microorganisms can be modified (e.g., engineered) totolerate the presence of additional microorganisms. In some embodiments,the microorganisms can be modified (e.g., engineered) to grow at adesired rate under desired conditions.

Culture Solutions

As detailed above, the methods described herein can be used to process afirst material (e.g., biomass), e.g., to change (e.g., lower) therecalcitrant level of the biomass, to produce a second material that canbe used as a substrate by microorganisms (e.g., bacteria, fungi (e.g.,yeasts and moulds), and/or algae), e.g., in or as a culture solution.Typically, culture solutions can be formulated based on their ability tosupport the growth of the selected microorganisms. In addition to thebiomass-based substrates generated herein, culture solutions can alsooptionally include an additional carbon source (e.g., glucose), water,salts, amino acids or an amino acid source. In some embodiments, culturesolutions can include a supplemental nitrogen source. The pH of theseculture solutions can be adapted to the requirement of the selectedmicroorganism. Culture solutions can also optionally include one or moreantibiotics to prevent contamination.

Certain culture solutions are commercially available, for example,commercially available growth medias include, Luria Bertani (LB) medium,terrific broth (TB) medium, yeast and mould (YM) broth (yeast extract 3g/L, malt extract 3 g/L, peptone 5 g/L, and dextrose 10 g/L and pH6.0-pH 8.0), YPG media (yeast extract, 3 g; mycological peptone, 5 g;D-glucose, 10 g per liter of water) and bacto peptone. Growth medias canbe purchased from commercial sources (e.g., Sigma Aldrich or Difco).Culture solutions useful in the present methods are provided in the art,for example, in Ramasamy et al., J. Appl. Biotechnol., 46:117-124, 1979,Young et al., Biotechnol Lett., 14:863-868, 1992, Anupama and Ravindra,Brazilian Archives or Biology and Biotechnol., 44:79-88, 2001, U.S. Pat.Nos. 3,627,095, 4,379,844, 4,447,530, 4,401,680, 4,526,721, 5,047,332,and 4,938,972. In some embodiments, any one of these commerciallyavailable or published culture solutions can be supplemented with thebiomass substrates generated herein.

In some embodiments, however, the use of commercially available mediaswill not be the most economically viable option. In such cases, culturesolutions can be prepared manually. In some embodiments, culturesolutions can contain, in addition to the biomass substrates generatedherein, per liter of water at pH 4-7.5: 1.88-2.357 g (NH₄)₂SO₄, 0.75-1.5g KH₂PO₄, 0.25-5 g MgSO₄.7H2O, 0.25-0.5 g FeS)₄.7H₂O, 0.25-0.5ZnSO₄.7H₂O, 0.1-1 ml trace element solution. In some embodiments, theculture solution can further include 114 mg boric acid, 480 mg ammoniummolybdate, 780 mg cupric sulphate, and 144 mg manganese chloride. Insome embodiments, the culture solution can further comprise 0.5 g yeastextract and can be used for the culture of yeast. In some embodiments,the culture solution can further comprise 1.0 g yeast extract and can beused for the culture of Zymomonas mobilis. In some embodiments, theculture solution can be adapted for the fermentation of ethanol and cancontain, in addition to the biomass substrates generated herein, perliter of water, sugars equivalent to 80-160 g glucose, 1 g KH₂PO₄, 1.5 gNH₄Cl, 0.16 g MgSO₄.7H2O, 0.08 g CaCl₂, and 1.0 g yeast extract.

In some embodiments, the selected microorganism can be a yeast and thegrowth media can contain, in addition to the biomass substratesgenerated herein, 1.7 g/L yeast nitrogen base, 2.27 g/L urea, 6.56 g/Lpeptone at pH 5.0.

In some embodiments, the selected microorganisms can be cultured in thepresence of a nitrogen source and/or an additional nitrogen source(e.g., when the desired products are proteins or amino acids). In suchcases, the nitrogen source can include any nitrogen source, for example,animal waste (e.g., poultry manure), human waste, inorganic nitrogensources, nitrite, nitrate, anhydrous ammonia, ammonium nitrate,diammonium phosphate, monoammonium phosphate, beef, or yeast extract. Insome embodiments, animal waste and human waste can be sterilized (e.g.,filtered or autoclaved) prior to use.

The selected microorganisms can be cultured on a small scale (e.g.,using standard laboratory equipment and methods known in the art) or ona large scale (e.g., using fermentation or industrial fermentationmethods). The choice of culture solution will depend on the desiredculture scale.

Culture Conditions

Cell culture conditions (e.g., temperature, pH and oxygen requirements)for most organisms are known in the art, and, if required, can be easilyoptimized as required. For example, culture conditions can be conductedbatchwise or continuously. The temperature used for cell culture can beselected according to the selected microorganisms so a to produceacceptable yields and substrate, particularly carbon, conversion ratios.Exemplary temperatures are within the range of 25-40° C. Similarly, thepH used for cell culture can be kept within a range at which maximumgrowth is exhibited for the selected microorganisms. Exemplary pH rangesare pH 5.0-8.0, e.g., pH 6.0-7.0. In addition, oxygenation levels can beadjusted to be maintained at a level that ensures optimal growth of theselected microorganism. For example, aerobic organisms can be culturedin an oxygenated environment. Alternatively, anaerobic organisms can becultured in an anaerobic environment.

Culture Methods

In some embodiments, the selected microorganisms can be cultured usingwithout the use of fermentation equipment. For example a firstlignocellulosic biomass material with a first recalcitrant level can beprocessed to produce a second material with an altered (e.g., lowered)recalcitrant level. This second material can then be used in abioconversion step to produce a product not present in the firstlignocellulosic biomass material. In some embodiments, this secondmaterial can be combined (e.g., in a liquid medium or culture) in a cellculture flask with one or more microorganisms under conditions suitablefor growth of the microorganisms and generation of the product. Theculture can then be incubated for a period of time sufficient togenerate the product.

In some embodiments, all cell culture equipment is sterilized or issterile prior to use.

Small Scale Methods

In some embodiments, the selected microorganisms can be cultured usingbench-top fermentation equipment. For example a first lignocellulosicbiomass material with a first recalcitrant level can be processed toproduce a second material with an altered (e.g., lowered) recalcitrantlevel. This second material can then be used in a bioconversion step toproduce a product not present in the first lignocellulosic biomassmaterial. In some embodiments, the second material can be combined withselected microorganisms and cultured in a bench top fermentor, e.g., aBraun (B. Braun Biotech, Aylesbury, Bucks) Biostat ER3 fermentor with aworking volume of 2.8 liters, in a growth media and under cultureconditions suitable for growth of the microorganisms and generation ofthe product. The process can then be maintained for a period of timesufficient to generate the product. Exemplary set points can include:temperature 20-45° C.; pH 3-9 (which can be maintained byautotitration); with defined agitation and air flow rates (e.g., about1000 rpm and 2 L/minute, respectively). In addition, foaming canoptionally be suppressed by the timed addition of an anti-foaming agent,e.g., a polypropylene glycol antifoam oil.

Large Scale Methods

In some embodiments, the selected microorganisms can be cultured usinglarge scale fermentation equipment (e.g., stirred tank bioreactorsand/or airlift bioreactors). For example a first lignocellulosic biomassmaterial with a first recalcitrant level can be processed to produce asecond material with an altered (e.g., lowered) recalcitrant level. Thissecond material can then be used in a bioconversion step to produce aproduct not present in the first lignocellulosic biomass material. Insome embodiments, the second material can be combined with selectedmicroorganisms and cultured, e.g., in a stirred tank bioreactor (e.g., a300 L stirred tank bioreactor). Alternatively or in addition, the secondmaterial can be combined with selected microorganisms and cultured in aairlift (pressure cycle) bioreactor (e.g., a 40,000 L airlift bioreactoras manufactured by RHM and ICI for the production of Quorn®). In bothcases, the second material can be combined with selected microorganismsin a culture solution and under culture conditions suitable for growthof the microorganisms and generation of the product. The process canthen be maintained for a period of time sufficient to generate theproduct.

In some embodiments, the selected microorganisms can be cultured usingfed-batch fermentation (e.g., fixed volume fed-batch or variable volumefed-batch) in which nutrients are added in a controlled manner inaccordance with the requirements of the culture solution (see FIG. 44and FIG. 45). In a fixed volume fed-batch fermentation process growthlimiting substrates are added to the culture solution in a highlyconcentrated form or a gas form that does not alter the volume of theculture solution. Once fermentation reaches a certain stage, a volume ofthe culture solution can optionally be removed and replaced with freshculture solution. In such a step, the volume of culture solution notremoved from the fermentor serves as the starter culture for the nextcycle and the removed volume contains the desired product. Such aprocess is referred to in the art as cyclic fed-batch culture for fixedvolume culture. One advantage of using cyclic fed-batch culture forfixed volume culture is that desired products can be obtained prior tothe end of the fermentation process. In addition, a cyclic fed-batchculture for fixed volume culture process can be continuous. In avariable volume fed-batch fermentation process, growth-limitingsubstrates are added as required to promote further growth of theculture in a concentration equal to the concentration of the startingculture. Consequently, the total volume of the culture increases. Thisprocess can be repeated until the volume of the culture reaches thecapacity of the fermentor. Larger fermentation tanks are advantageous inthis method as such tanks accommodate larger volumes of culturesolution. The desired products can then be obtained from the culturesolution, e.g., at the end of the fermentation process. Both thesefed-batch processes allow optimal yields and productivities. In someembodiments, the process can include providing continuously oxygenatedwater, e.g., using an air-lift fermentation system.

Fed-batch processes are also described in European Patent ApplicationNo. 533039.

Following fermentation, the selected microorganism and/or product can beharvested and optionally isolated and/or purified. Methods forharvesting microorganisms from culture solutions include, for example,centrifugation and/or filtration.

Further Processing for Food Products

Cultures for use, e.g., as ingestible foods for animals and/or humanscan be further processed, e.g., using the methods discloses in U.S. Pat.Nos. 5,935,841; 6,270,816; 5,980,958; and 3,809,614. Alternatively or inaddition, a harvested organism can be treated to reduce its nucleic acidcontent, e.g., using the process of UK Patent No. 1,440,642; separated,if desired, e.g., using the process of UK Patent No. 1,473,654, or byfiltration or centrifugation; and its palatability can be modified,e.g., using the procedures of UK Patent Nos. 1,508,635; 1,502,455;1,496,113; and/or 1,587,828.

Humans do not possess the enzyme uricase to catalyze the conversion ofuric acid to the more soluble allantoin. Consumption of microbial cells,which contain high levels of nucleic acid, can, therefore, lead toelevated levels of uric acid and complications associated therewith inhumans. In some embodiments, therefore, nucleic acids can be removed orreduced from samples containing microbial cells or food products derivedfrom microbial cells (e.g., proteins, fats and oils, and carbohydrates)prior to consumption by humans, for example, using methods as describedby Lawford and Lewis (U.S. Pat. No. 4,330,464). In some embodiments,nucleic acids can be removed or reduced from samples containingmicrobial cells or food products derived from microbial cells (e.g.,proteins, fats and oils, and carbohydrates) prior to consumption byhumans, e.g., using the methods described in U.S. Pat. No. 6,270,816.For example, microbial cells can be killed and the nucleic acidsimultaneously reduced by rapidly heating the culture solution to atleast 60° C. This process can be used to promote loss of cell viabilityand reduction of a portion of cellular nucleic acid (e.g., DNA and RNA)into the supernatant. Following heating, the culture solution can becentrifuged and rinsed to remove the nucleic acids.

In some embodiments, the protein:RNA ratio for protein in a sample forhuman consumption should be at least 12:1. In some embodiments, thetotal nucleic acid content of a sample for human consumption can bereduced to about 2% (e.g., 2%, less than 2%, 0.1-2.0%, 0.1-1.5%, 0.1-1%,0.1-0.5%, 0.1-0.3%, 0.1%) of the dry weight of the sample.

In some embodiments, nutritional and/or toxicological evaluations ofsamples containing microbial cells or food products derived frommicrobial cells (e.g., proteins, fats and oils, and carbohydrates) canbe performed prior to ingestion by animals (e.g., for each targetspecies).

In some embodiments, microbial proteins can be dried, lyophilized, or insolution, and can be present in an isolated form or in the presence ofone or more additional food sources.

In some embodiments, samples containing microbial cells or food productsderived from microbial cells (e.g., proteins, fats and oils, andcarbohydrates) can be formulated as edible gels. Gel quality can beassessed using Strain and stress tests, e.g., using the torsiontechnique of Wu et al., J. Tex. Studies, 16: 53-74 (1985), or with aRheo Tex model gelometer AP-83 (Sun Sciences Co. Seattle, Wash., USA).In general, values of strain (elastic component) greater than 1.9 to 2.0and stress values of 30-35 kPa are a reliable indication of gelstrength. In some embodiments, samples containing microbial cells orfood products derived from microbial cells (e.g., proteins, fats andoils, and carbohydrates) can be flavored and/or colored, e.g., toincrease palatability for the target species.

In some embodiments, samples containing microbial cells or food productsderived from microbial cells (e.g., proteins, fats and oils, andcarbohydrates) can be used as or in the generation of meat analogues.“Meat analogue” is an industrial term for meat substitutes or syntheticmeats made primarily from non-animal source, e.g., plant proteins.

In some embodiments, the health and nutritional values of the foodproducts derived from microbial cells (e.g., proteins, fats and oils,and carbohydrates) described herein are considered prior to consumptionby animals and/or humans.

Food Formulations

In some embodiments, the food products described herein can be used asor in the production of food products (e.g., solid or liquid foodproducts). In some embodiments, the food products can be used alone orcan be combined. In some embodiments, the food products can be combinedwith texturizing materials (e.g., wheat protein). In some embodiments,the food products disclosed herein can be formulated as meatalternatives (see, e.g., Quorn®, manufactured by Marlow Foods, UK). Insome embodiments, the food products disclosed herein can be combinedwith other proteins, protein sources, or foods, e.g., mycoprotein,textured vegetable protein, tofu, tempeh, miso, soya products, and/orwheat protein.

In some embodiments, any of the products and co-products describedherein can be combined with a flavorings and/or colorings, for example,fine chemical flavors and aromas.

Process Water

In the processes disclosed herein, whenever water is used in anyprocess, it may be grey water, e.g., municipal grey water, or blackwater. In some embodiments, the grey or black water is sterilized priorto use. Sterilization may be accomplished by any desired technique, forexample by irradiation, steam, or chemical sterilization.

EXAMPLES

The following Examples are intended to illustrate, and do not limit theteachings of this disclosure.

Example 1 Preparation of Fibrous Material from Polycoated Paper

A 1500 pound skid of virgin, half-gallon juice cartons made ofun-printed polycoated white Kraft board having a bulk density of 20lb/ft³ was obtained from International Paper. Each carton was foldedflat, and then fed into a 3 hp Flinch Baugh shredder at a rate ofapproximately 15 to 20 pounds per hour. The shredder was equipped withtwo 12 inch rotary blades, two fixed blades and a 0.30 inch dischargescreen. The gap between the rotary and fixed blades was adjusted to 0.10inch. The output from the shredder resembled confetti having a width ofbetween 0.1 inch and 0.5 inch, a length of between 0.25 inch and 1 inchand a thickness equivalent to that of the starting material (about 0.075inch).

The confetti-like material was fed to a Munson rotary knife cutter,Model SC30. Model SC30 is equipped with four rotary blades, four fixedblades, and a discharge screen having ⅛ inch openings. The gap betweenthe rotary and fixed blades was set to approximately 0.020 inch. Therotary knife cutter sheared the confetti-like pieces across theknife-edges, tearing the pieces apart and releasing a fibrous materialat a rate of about one pound per hour. The fibrous material had a BETsurface area of 0.9748 m²/g+/−0.0167 m²/g, a porosity of 89.0437 percentand a bulk density (@0.53 psia) of 0.1260 g/mL. An average length of thefibers was 1.141 mm and an average width of the fibers was 0.027 mm,giving an average L/D of 42:1. A scanning electron micrograph of thefibrous material is shown in FIG. 26 at 25× magnification.

Example 2 Preparation of Fibrous Material from Bleached Kraft Board

A 1500 pound skid of virgin bleached white Kraft board having a bulkdensity of 30 lb/ft³ was obtained from International Paper. The materialwas folded flat, and then fed into a 3 hp Flinch Baugh shredder at arate of approximately 15 to 20 pounds per hour. The shredder wasequipped with two 12 inch rotary blades, two fixed blades and a 0.30inch discharge screen. The gap between the rotary and fixed blades wasadjusted to 0.10 inch. The output from the shredder resembled confettihaving a width of between 0.1 inch and 0.5 inch, a length of between0.25 inch and 1 inch and a thickness equivalent to that of the startingmaterial (about 0.075 inch). The confetti-like material was fed to aMunson rotary knife cutter, Model SC30. The discharge screen had ⅛ inchopenings. The gap between the rotary and fixed blades was set toapproximately 0.020 inch. The rotary knife cutter sheared theconfetti-like pieces, releasing a fibrous material at a rate of aboutone pound per hour. The fibrous material had a BET surface area of1.1316 m²/g+/−0.0103 m²/g, a porosity of 88.3285 percent and a bulkdensity (@0.53 psia) of 0.1497 g/mL. An average length of the fibers was1.063 mm and an average width of the fibers was 0.0245 mm, giving anaverage L/D of 43:1. A scanning electron micrograph of the fibrousmaterial is shown in FIG. 27 at 25× magnification.

Example 3 Preparation of Twice Sheared Fibrous Material from BleachedKraft Board

A 1500 pound skid of virgin bleached white Kraft board having a bulkdensity of 30 lb/ft³ was obtained from International Paper. The materialwas folded flat, and then fed into a 3 hp Flinch Baugh shredder at arate of approximately 15 to 20 pounds per hour. The shredder wasequipped with two 12 inch rotary blades, two fixed blades and a 0.30inch discharge screen. The gap between the rotary and fixed blades wasadjusted to 0.10 inch. The output from the shredder resembled confetti(as above). The confetti-like material was fed to a Munson rotary knifecutter, Model SC30. The discharge screen had 1/16 inch openings. The gapbetween the rotary and fixed blades was set to approximately 0.020 inch.The rotary knife cutter the confetti-like pieces, releasing a fibrousmaterial at a rate of about one pound per hour. The material resultingfrom the first shearing was fed back into the same setup described aboveand sheared again. The resulting fibrous material had a BET surface areaof 1.4408 m²/g+/−0.0156 m²/g, a porosity of 90.8998 percent and a bulkdensity (@0.53 psia) of 0.1298 g/mL. An average length of the fibers was0.891 mm and an average width of the fibers was 0.026 mm, giving anaverage L/D of 34:1. A scanning electron micrograph of the fibrousmaterial is shown in FIG. 28 at 25× magnification.

Example 4 Preparation of Thrice Sheared Fibrous Material from BleachedKraft Board

A 1500 pound skid of virgin bleached white Kraft board having a bulkdensity of 30 lb/ft³ was obtained from International Paper. The materialwas folded flat, and then fed into a 3 hp Flinch Baugh shredder at arate of approximately 15 to 20 pounds per hour. The shredder wasequipped with two 12 inch rotary blades, two fixed blades and a 0.30inch discharge screen. The gap between the rotary and fixed blades wasadjusted to 0.10 inch. The output from the shredder resembled confetti(as above). The confetti-like material was fed to a Munson rotary knifecutter, Model SC30. The discharge screen had ⅛ inch openings. The gapbetween the rotary and fixed blades was set to approximately 0.020 inch.The rotary knife cutter sheared the confetti-like pieces across theknife-edges. The material resulting from the first shearing was fed backinto the same setup and the screen was replaced with a 1/16 inch screen.This material was sheared. The material resulting from the secondshearing was fed back into the same setup and the screen was replacedwith a 1/32 inch screen. This material was sheared. The resultingfibrous material had a BET surface area of 1.6897 m²/g+/−0.0155 m²/g, aporosity of 87.7163 percent and a bulk density (@0.53 psia) of 0.1448g/mL. An average length of the fibers was 0.824 mm and an average widthof the fibers was 0.0262 mm, giving an average L/D of 32:1. A scanningelectron micrograph of the fibrous material is shown in FIG. 29 at 25×magnification.

Example 5 Preparation of Densified Fibrous Material from Bleached KraftBoard without Added Binder

Fibrous material was prepared according to Example 2. Approximately 1 lbof water was sprayed onto each 10 lb of fibrous material. The fibrousmaterial was densified using a California Pellet Mill 1100 operating at75° C. Pellets were obtained having a bulk density ranging from about 7lb/ft³ to about 15 lb/ft³.

Example 6 Preparation of Densified Fibrous Material from Bleached KraftBoard with Binder

Fibrous material was prepared according to Example 2.

A 2 weight percent stock solution of POLYOX™ WSR N10 (polyethyleneoxide) was prepared in water.

Approximately 1 lb of the stock solution was sprayed onto each 10 lb offibrous material. The fibrous material was densified using a CaliforniaPellet Mill 1100 operating at 75° C. Pellets were obtained having a bulkdensity ranging from about 15 lb/ft³ to about 40 lb/ft³.

Example 7 Reducing the Molecular Weight of Cellulose in Fibrous KraftPaper by Gamma Radiation with Minimum Oxidation

Fibrous material is prepared according to Example 4, and then densifiedaccording to Example 5.

The densified pellets are placed in a glass ampoule having a maximumcapacity of 250 mL. The glass ampoule is evacuated under high vacuum(10⁻⁵ ton) for 30 minutes, and then back-filled with argon gas. Theampoule is sealed under argon. The pellets in the ampoule are irradiatedwith gamma radiation for about 3 hours at a dose rate of about 1 Mradper hour to provide an irradiated material in which the cellulose has alower molecular weight than the fibrous Kraft starting material.

Example 8 Reducing the Molecular Weight of Cellulose in Fibrous KraftPaper by Gamma Radiation with Maximum Oxidation

Fibrous material is prepared according to Example 4, and then densifiedaccording to Example 5.

The densified pellets are placed in a glass ampoule having a maximumcapacity of 250 mL. The glass ampoule is sealed under an atmosphere ofair. The pellets in the ampoule are irradiated with gamma radiation forabout 3 hours at a dose rate of about 1 Mrad per hour to provide anirradiated material in which the cellulose has a lower molecular weightthan the fibrous Kraft starting material.

Example 9 Methods of Determining Molecular Weight of Cellulosic andLignocellulosic Materials by Gel Permeation Chromatography

Cellulosic and lignocellulosic materials for analysis were treatedaccording to Example 4. Sample materials presented in the followingtables include Kraft paper (P), wheat straw (WS), alfalfa (A), andswitchgrass (SG). The number “132” of the Sample ID refers to theparticle size of the material after shearing through a 1/32 inch screen.The number after the dash refers to the dosage of radiation (MRad) and“US” refers to ultrasonic treatment. For example, a sample ID “P132-10”refers to Kraft paper that has been sheared to a particle size of 132mesh and has been irradiated with 10 MRad.

TABLE 1 Peak Average Molecular Weight of Irradiated Kraft Paper SampleSample Dosage¹ Average MW ± Source ID (MRad) Ultrasound² Std Dev. KraftPaper P132 0 No 32853 ± 10006 P132-10 10 ″  61398 ± 2468** P132-100 100″ 8444 ± 580  P132-181 181 ″ 6668 ± 77  P132-US 0 Yes 3095 ± 1013 **Lowdoses of radiation appear to increase the molecular weight of somematerials ¹Dosage Rate = 1 MRad/hour ²Treatment for 30 minutes with 20kHz ultrasound using a 1000 W horn under re-circulating conditions withthe material dispersed in water.

TABLE 2 Peak Average Molecular Weight of Irradiated Materials SamplePeak Dosage¹ Average MW ± ID # (MRad) Ultrasound² Std Dev. WS132 1  0 No1407411 ± 175191 2 ″ ″ 39145 ± 3425 3 ″ ″ 2886 ± 177 WS132-10* 1 10 ″26040 ± 3240 WS132-100* 1 100  ″ 23620 ± 453  A132 1  0 ″ 1604886 ±151701 2 ″ ″ 37525 ± 3751 3 ″ ″ 2853 ± 490 A132-10* 1 10 ″ 50853 ± 16652 ″ ″ 2461 ± 17  A132-100* 1 100  ″ 38291 ± 2235 2 ″ ″ 2487 ± 15  SG1321  0 ″ 1557360 ± 83693  2 ″ ″ 42594 ± 4414 3 ″ ″ 3268 ± 249 SG132-10* 110 ″ 60888 ± 9131 SG132-100* 1 100  ″ 22345 ± 3797 SG132-10-US 1 10 Yes 86086 ± 43518 2 ″ ″ 2247 ± 468 SG132-100-US 1 100  ″  4696 ± 1465*Peaks coalesce after treatment **Low doses of radiation appear toincrease the molecular weight of some materials ¹Dosage Rate = 1MRad/hour ²Treatment for 30 minutes with 20 kHz ultrasound using a 1000W horn under re-circulating conditions with the material dispersed inwater.

Gel Permeation Chromatography (GPC) is used to determine the molecularweight distribution of polymers. During GPC analysis, a solution of thepolymer sample is passed through a column packed with a porous geltrapping small molecules. The sample is separated based on molecularsize with larger molecules eluting sooner than smaller molecules. Theretention time of each component is most often detected by refractiveindex (RI), evaporative light scattering (ELS), or ultraviolet (UV) andcompared to a calibration curve. The resulting data is then used tocalculate the molecular weight distribution for the sample.

A distribution of molecular weights rather than a unique molecularweight is used to characterize synthetic polymers. To characterize thisdistribution, statistical averages are utilized. The most common ofthese averages are the “number average molecular weight” (M_(n)) and the“weight average molecular weight” (M_(w)). Methods of calculating thesevalues are described in Example 9 of PCT/US/2007/022719.

The polydispersity index or PI is defined as the ratio of M_(w)/M_(n).The larger the PI, the broader or more disperse the distribution. Thelowest value that a PI can be is 1. This represents a monodispersesample; that is, a polymer with all of the molecules in the distributionbeing the same molecular weight.

The peak molecular weight value (Mp) is another descriptor defined asthe mode of the molecular weight distribution. It signifies themolecular weight that is most abundant in the distribution. This valuealso gives insight to the molecular weight distribution.

Most GPC measurements are made relative to a different polymer standard.The accuracy of the results depends on how closely the characteristicsof the polymer being analyzed match those of the standard used. Theexpected error in reproducibility between different series ofdeterminations, calibrated separately, is ca. 5-10% and ischaracteristic of the limited precision of GPC determinations.Therefore, GPC results are most useful when a comparison between themolecular weight distributions of different samples is made during thesame series of determinations.

The lignocellulosic samples required sample preparation prior to GPCanalysis. First, a saturated solution (8.4% by weight) of lithiumchloride (LiCl) was prepared in dimethyl acetamide (DMAc). Approximately100 mg of each sample was added to approximately 10 g of a freshlyprepared saturated LiCl/DMAc solution, and the mixtures were heated toapproximately 150° C.-170° C. with stirring for 1 hour. The resultingsolutions were generally light- to dark-yellow in color. The temperatureof the solutions was decreased to approximately 100° C. and heated foran additional 2 hours. The temperature of the solutions was thendecreased to approximately 50° C. and the sample solutions were heatedfor approximately 48 to 60 hours. Of note, samples irradiated at 100MRad were more easily solubilized as compared to their untreatedcounterpart. Additionally, the sheared samples (denoted by the number132) had slightly lower average molecular weights as compared with uncutsamples.

The resulting sample solutions were diluted 1:1 using DMAc as solventand were filtered through a 0.45 μm PTFE filter. The filtered samplesolutions were then analyzed by GPC. The peak average molecular weight(Mp) of the samples, as determined by Gel Permeation Chromatography(GPC), are summarized in Tables 1 and 2, as above, under analysisconditions set forth in Table 3. Each sample was prepared in duplicateand each preparation of the sample was analyzed in duplicate (twoinjections) for a total of four injections per sample. The EasiCalpolystyrene standards PS1A and PS1B were used to generate a calibrationcurve for the molecular weight scale from about 580 to 7,500,00 Daltons.

TABLE 3 GPC Analysis Conditions Instrument: Waters Alliance GPC 2000Columns (3): Plgel 10μ Mixed-B S/N's: 10M-MB-148-83; 10M- MB-148-84;10M-MB-174-129 Mobile Phase (solvent): 0.5% LiCl in DMAc (1.0 mL/min.)Column/Detector Temperature: 70° C. Injector Temperature: 70° C. SampleLoop Size: 323.5 μL

Example 10 Determining Crystallinity of Irradiated Material by X-RayDiffraction

X-ray diffraction (XRD) is a method by which a crystalline sample isirradiated with monoenergetic x-rays. The interaction of the latticestructure of the sample with these x-rays is recorded and providesinformation about the crystalline structure being irradiated. Theresulting characteristic “fingerprint” allows for the identification ofthe crystalline compounds present in the sample. Using a whole-patternfitting analysis (the Rietvelt Refinement), it is possible to performquantitative analyses on samples containing more than one crystallinecompound.

Each sample was placed on a zero background holder and placed in aPhillips PW1800 diffractometer using Cu radiation. Scans were then runover the range of 5° to 50° with a step size of 0.05° and a countingtime of 2 hours each.

Once the diffraction patterns were obtained, the phases were identifiedwith the aid of the Powder Diffraction File published by theInternational Centre for Diffraction Data. In all samples thecrystalline phase identified was cellulose—Ia, which has a triclinicstructure.

The distinguishing feature among the 20 samples is the peak breadth,which is related to the crystallite domain size. The experimental peakbreadth was used to compute the domain size and percent crystallinity,which are reported in Table 4.

TABLE 4 XRD Data Including Domain Size and % Crystallinity Domain %Sample ID Size (Å) Crystallinity P132 55 55 P132-10 46 58 P132-100 50 55P132-181 48 52 P132-US 26 40 A132 28 42 A132-10 26 40 A132-100 28 35WS132 30 36 WS132-10 27 37 WS132-100 30 41 SG132 29 40 SG132-10 28 38SG132-100 28 37 SG132-10-US 25 42 SG132-100-US 21 34

Percent crystallinity (X_(c) %) is measured as a ratio of thecrystalline area to the total area under the x-ray diffraction peaks,and equals 100%×(A_(c)/(A_(a)+A_(c)), where

-   -   A_(c)=Area of crystalline phase    -   A_(a)=Area of amorphous phase    -   X_(c)=Percent of crystallinity

To determine the percent crystallinity for each sample it was necessaryto first extract the amount of the amorphous phase. This is done byestimating the area of each diffraction pattern that can be attributedto the crystalline phase (represented by the sharper peaks) and thenon-crystalline phase (represented by the broad humps beneath thepattern and centered at 22° and 38°).

A systematic process was used to minimize error in these calculationsdue to broad crystalline peaks as well as high background intensity,First, a linear background was applied and then removed. Second, twoGaussian peaks centered at 22° and 38° with widths of 10-12° each werefitted to the humps beneath the crystalline peaks. Third, the areabeneath the two broad Gaussian peaks and the rest of the pattern weredetermined. Finally, percent crystallinity was calculated by dividingthe area beneath the crystalline peak by the total intensity (afterbackground subtraction). Domain size and % crystallinity of the samplesas determined by X-ray diffraction (XRD) are presented in Table 4,above.

Example 11 Porosimetry Analysis

Mercury pore size and pore volume analysis (Table 5) is based on forcingmercury (a non-wetting liquid) into a porous structure under tightlycontrolled pressures. Since mercury does not wet most substances andwill not spontaneously penetrate pores by capillary action, it must beforced into the voids of the sample by applying external pressure. Thepressure required to fill the voids is inversely proportional to thesize of the pores. Only a small amount of force or pressure is requiredto fill large voids, whereas much greater pressure is required to fillvoids of very small pores.

TABLE 5 Pore Size and Volume Distribution by Mercury Porosimetry MedianMedian Average Bulk Total Total Pore Pore Pore Density ApparentIntrusion Pore Diameter Diameter Diameter @ 0.50 (skeletal) SampleVolume Area (Volume) (Area) (4 V/A) psia Density Porosity ID (mL/g)(m²/g) (μm) (μm) (μm) (g/mL) (g/mL) (%) P132 6.0594 1.228 36.225013.7278 19.7415 0.1448 1.1785 87.7163 P132- 5.5436 1.211 46.3463 4.564618.3106 0.1614 1.5355 89.4875 10 P132- 5.3985 0.998 34.5235 18.200521.6422 0.1612 1.2413 87.0151 100 P132- 3.2866 0.868 25.3448 12.241015.1509 0.2497 1.3916 82.0577 181 P132- 6.0005 14.787 98.3459 0.00551.6231 0.1404 0.8894 84.2199 US A132 2.0037 11.759 64.6308 0.0113 0.68160.3683 1.4058 73.7990 A132- 1.9514 10.326 53.2706 0.0105 0.7560 0.37681.4231 73.5241 10 A132- 1.9394 10.205 43.8966 0.0118 0.7602 0.37601.3889 72.9264 100 SG132 2.5267 8.265 57.6958 0.0141 1.2229 0.31191.4708 78.7961 SG132- 2.1414 8.643 26.4666 0.0103 0.9910 0.3457 1.331574.0340 10 SG132- 2.5142 10.766 32.7118 0.0098 0.9342 0.3077 1.359077.3593 100 SG132- 4.4043 1.722 71.5734 1.1016 10.2319 0.1930 1.288385.0169 10-US SG132- 4.9665 7.358 24.8462 0.0089 2.6998 0.1695 1.073184.2010 100-US WS132 2.9920 5.447 76.3675 0.0516 2.1971 0.2773 1.627982.9664 WS132- 3.1138 2.901 57.4727 0.3630 4.2940 0.2763 1.9808 86.048410 WS132- 3.2077 3.114 52.3284 0.2876 4.1199 0.2599 1.5611 83.3538 100

The AutoPore® 9520, a device for determining pore density, can attain amaximum pressure of 414 MPa or 60,000 psia. There are four low pressurestations for sample preparation and collection of macropore data from0.2 psia to 50 psia. There are two high pressure chambers that collectdata from 25 psia to 60,000 psia. The sample is placed in a bowl-likeapparatus called a penetrometer, which is bonded to a glass capillarystem with a metal coating. As mercury invades the voids in and aroundthe sample, it moves down the capillary stem. The loss of mercury fromthe capillary stem results in a change in the electrical capacitance.The change in capacitance during the experiment is converted to volumeof mercury based on the stem volume of the penetrometer in use. Avariety of penetrometers with different bowl (sample) sizes andcapillaries are available to accommodate most sample sizes andconfigurations. Table 6 below defines some of the key parameterscalculated for each sample.

TABLE 6 Definition of Parameters Parameter Description Total IntrusionThe total volume of mercury intruded during an Volume: experiment. Thiscan include interstitial filling between small particles, porosity ofsample, and compression volume of sample. Total Pore Area: The totalintrusion volume converted to an area assuming cylindrical shaped pores.Median Pore The size at the 50^(th) percentile on the cumulativeDiameter (volume): volume graph. Median Pore The size at the 50^(th)percentile on the cumulative Diameter (area): area graph. Average PoreThe total pore volume divided by the total pore area Diameter: (4 V/A).Bulk Density: The mass of the sample divided by the bulk volume. Bulkvolume is determined at the filling pressure, typically 0.5 psia.Apparent Density: The mass of sample divided by the volume of samplemeasured at highest pressure, typically 60,000 psia. Porosity: (BulkDensity/Apparent Density) × 100%

Example 12 Particle Size Analysis

The technique of particle sizing by static light scattering is based onMie theory (which also encompasses Fraunhofer theory). Mie theorypredicts the intensity vs. angle relationship as a function of the sizefor spherical scattering particles provided that other system variablesare known and held constant. These variables are the wavelength ofincident light and the relative refractive index of the sample material.Application of Mie theory provides the detailed particle sizeinformation. Table 7 summarizes particle size using median diameter,mean diameter, and modal diameter as parameters.

TABLE 7 Particle Size by Laser Light Scattering (Dry Sample Dispersion)Median Diameter Mean Diameter Modal Diameter Sample ID (μm) (μm) (μm)A132 380.695 418.778 442.258 A132-10 321.742 366.231 410.156 A132-100301.786 348.633 444.169 SG132 369.400 411.790 455.508 SG132-10 278.793325.497 426.717 SG132-100 242.757 298.686 390.097 WS132 407.335 445.618467.978 WS132-10 194.237 226.604 297.941 WS132-100 201.975 236.037307.304

Particle size was determined by laser light scattering (dry sampledispersion) using a Malvern Mastersizer 2000 using the followingconditions:

-   -   Feed Rate: 35%    -   Disperser Pressure: 4 Bar    -   Optical Model: (2.610, 1.000i), 1.000

An appropriate amount of sample was introduced onto a vibratory tray.The feed rate and air pressure were adjusted to ensure that theparticles were properly dispersed. The key component is selecting an airpressure that will break up agglomerations, but does not compromise thesample integrity. The amount of sample needed varies depending on thesize of the particles. In general, samples with fine particles requireless material than samples with coarse particles.

Example 13 Surface Area Analysis

Surface area of each sample was analyzed using a Micromeritics ASAP 2420Accelerated Surface Area and Porosimetry System. The samples wereprepared by first degassing for 16 hours at 40° C. Next, free space(both warm and cold) with helium is calculated and then the sample tubeis evacuated again to remove the helium. Data collection begins afterthis second evacuation and consists of defining target pressures whichcontrols how much gas is dosed onto the sample. At each target pressure,the quantity of gas adsorbed and the actual pressure are determined andrecorded. The pressure inside the sample tube is measured with apressure transducer. Additional doses of gas will continue until thetarget pressure is achieved and allowed to equilibrate. The quantity ofgas adsorbed is determined by summing multiple doses onto the sample.The pressure and quantity define a gas adsorption isotherm and are usedto calculate a number of parameters, including BET surface area (Table8).

TABLE 8 Summary of Surface Area by Gas Adsorption Single point surfacearea BET Surface Sample ID @ P/Po = (m2/g) Area (m²/g) P132 0.2503877711.5253 1.6897 P132-10 @ P/Po = 1.0212 1.2782 0.239496722 P132-100 @ P/Po= 1.0338 1.2622 0.240538100 P132-181 @ P/Po = 0.5102 0.6448 0.239166091P132-US @ P/Po = 1.0983 1.6793 0.217359072 A132 @ P/Po = 0.5400 0.76140.240040610 A132-10 @ P/Po = 0.5383 0.7212 0.211218936 A132-100 @ P/Po =0.4258 0.5538 0.238791097 SG132 @ P/Po = 0.6359 0.8350 0.237989353SG132-10 @ P/Po = 0.6794 0.8689 0.238576905 SG132-100 @ P/Po = 0.55180.7034 0.241960361 SG132-10-US @ P/Po = 0.5693 0.7510 0.225692889SG132-100-US @ P/Po = 1.0983 1.4963 0.225935246 WS132 @ P/Po = 0.65820.8663 0.237823664 WS132-10 @ P/Po = 0.6191 0.7912 0.238612476 WS132-100@ P/Po = 0.6255 0.8143 0.238398832

The BET model for isotherms is a widely used theory for calculating thespecific surface area. The analysis involves determining the monolayercapacity of the sample surface by calculating the amount required tocover the entire surface with a single densely packed layer of krypton.The monolayer capacity is multiplied by the cross sectional area of amolecule of probe gas to determine the total surface area. Specificsurface area is the surface area of the sample aliquot divided by themass of the sample.

Example 14 Fiber Length Determination

Fiber length distribution testing was performed in triplicate on thesamples submitted using the Techpap MorFi LB01 system. The averagelength and width are reported in Table 9.

TABLE 9 Summary of Lignocellulosic Fiber Length and Width Data AverageStatistically Arithmetic Length Corrected Average Average Weighted inLength Weighted in Width Sample ID (mm) Length (mm) Length (mm) (μm)P132-10 0.484 0.615 0.773 24.7 P132-100 0.369 0.423 0.496 23.8 P132-1810.312 0.342 0.392 24.4 A132-10 0.382 0.423 0.650 43.2 A132-100 0.3620.435 0.592 29.9 SG132-10 0.328 0.363 0.521 44.0 SG132-100 0.325 0.3510.466 43.8 WS132-10 0.353 0.381 0.565 44.7 WS132-100 0.354 0.371 0.53645.4

Example 15 Ultrasonic Treatment of Irradiated and Un-irradiatedSwitchgrass

Switchgrass was sheared according to Example 4. The switchgrass wastreated by ultrasound alone or irradiation with 10 Mrad or 100 Mrad ofgamma rays, and then sonicated. The resulting materials correspond toG132-BR (un-irradiated), G132-10-BR (10 Mrad and sonication) andG132-100-BR (100 Mrad and sonication), as presented in Table 1.Sonication was performed on each sample for 30 minutes using 20 kHzultrasound from a 1000 W horn under re-circulating conditions. Eachsample was dispersed in water at a concentration of about 0.10 g/mL.

FIGS. 30 and 31 show the apparatus used for sonication. Apparatus 500includes a converter 502 connected to booster 504 communicating with ahorn 506 fabricated from titanium or an alloy of titanium. The horn,which has a seal 510 made from VITON® about its perimeter on itsprocessing side, forms a liquid tight seal with a processing cell 508.The processing side of the horn is immersed in a liquid, such as water,that has dispersed therein the sample to be sonicated. Pressure in thecell is monitored with a pressure gauge 512. In operation, each sampleis moved by pump 517 from tank 516 through the processing cell and issonicated. After, sonication, the sample is captured in tank 520. Theprocess can be reversed in that the contents of tank 520 can be sentthrough the processing cell and captured in tank 516. This process canbe repeated a number of times until a desired level of processing isdelivered to the sample.

Example 16 Scanning Electron Micrographs of Un-irradiated Switchgrass inComparison to Irradiated and Irradiated and Sonicated Switchgrass

Switchgrass samples for the scanning electron micrographs were appliedto carbon tape and gold sputter coated (70 seconds). Images were takenwith a JEOL 6500 field emission scanning electron microscope.

FIG. 32 is a scanning electron micrograph at 1000× magnification of afibrous material produced from shearing switchgrass on a rotary knifecutter, and then passing the sheared material through a 1/32 inchscreen.

FIGS. 33 and 34 are scanning electron micrographs of the fibrousmaterial of FIG. 32 after irradiation with 10 Mrad and 100 Mrad gammarays, respectively, at 1000× magnification.

FIG. 35 is a scanning electron micrograph of the fibrous material ofFIG. 32 after irradiation with 10 Mrad and sonication at 1000×magnification.

FIG. 36 is a scanning electron micrograph of the fibrous material ofFIG. 32 after irradiation with 100 Mrad and sonication at 1000×magnification.

Example 17 Infrared Spectrum of Irradiated Kraft Paper in Comparison toUn-Irradiated Kraft Paper

FT-IR analysis was performed using standard methodology on aNicolet/Impact 400. The results indicate that all samples reported inTable 1 are consistent with a cellulose-based material.

FIG. 37 is an infrared spectrum of Kraft board paper sheared accordingto Example 4, while FIG. 38 is an infrared spectrum of the Kraft paperof FIG. 38 after irradiation with 100 Mrad of gamma radiation. Theirradiated sample shows an additional peak in region A (centered about1730 cm⁻¹) that is not found in the un-irradiated material.

Example 18 Combination of Electron Beam and Sonication Pretreatment

Switchgrass is used as the feedstock and is sheared with a Munson rotaryknife cutter into a fibrous material. The fibrous material is thenevenly distributed onto an open tray composed of tin with an area ofgreater than about 500 in². The fibrous material is distributed so thatit has a depth of about 1-2 inches in the open tray. The fibrousmaterial can be distributed in plastic bags at lower doses ofirradiation (under 10 MRad), and left uncovered on the metal tray athigher doses of radiation.

Separate samples of the fibrous material are then exposed to successivedoses of electron beam radiation to achieve a total dose of 1 Mrad, 2Mrad, 3, Mrad, 5 Mrad, 10 Mrad, 50 Mrad, and 100 Mrad. Some samples aremaintained under the same conditions as the remaining samples, but arenot irradiated, to serve as controls. After cooling, the irradiatedfibrous material is sent on for further processing through a sonicationdevice.

The sonication device includes a converter connected to boostercommunicating with a horn fabricated from titanium or an alloy oftitanium. The horn, which has a seal made from VITON® about itsperimeter on its processing side, forms a liquid tight seal with aprocessing cell. The processing side of the horn is immersed in aliquid, such as water, into which the irradiated fibrous material to besonicated is immersed. Pressure in the cell is monitored with a pressuregauge. In operation, each sample is moved by pump through the processingcell and is sonicated.

To prepare the irradiated fibrous material for sonication, theirradiated fibrous material is removed from any container (e.g., plasticbags) and is dispersed in water at a concentration of about 0.10 g/mL.Sonication is performed on each sample for 30 minutes using 20 kHzultrasound from a 1000 W horn under re-circulating conditions. Aftersonication, the irradiated fibrous material is captured in a tank. Thisprocess can be repeated a number of times until a desired level ofprocessing is achieved based on monitoring the structural changes in theswitchgrass. Again, some irradiated samples are kept under the sameconditions as the remaining samples, but are not sonicated, to serve ascontrols. In addition, some samples that were not irradiated aresonicated, again to serve as controls. Thus, some controls are notprocessed, some are only irradiated, and some are only sonicated.

Example 19 Microbial Testing of Pretreated Biomass

Specific lignocellulosic materials pretreated as described herein areanalyzed for toxicity to common strains of yeast and bacteria used inthe biofuels industry for the fermentation step in ethanol production.Additionally, sugar content and compatibility with cellulase enzymes areexamined to determine the viability of the treatment process. Testing ofthe pretreated materials is carried out in two phases as follows.

I. Toxicity and Sugar Content

Toxicity of the pretreated grasses and paper feedstocks is measured inyeast Saccharomyces cerevisiae (wine yeast) and Pichia stipitis (ATCC66278) as well as the bacteria Zymomonas mobilis (ATCC 31821) andClostridium thermocellum (ATCC 31924). A growth study is performed witheach of the organisms to determine the optimal time of incubation andsampling.

Each of the feedstocks is then incubated, in duplicate, with S.cerevisiae, P. stipitis, Z. mobilis, and C. thermocellum in a standardmicrobiological medium for each organism. YM broth is used for the twoyeast strains, S. cerevisiae and P. stipitis. RM medium is used for Z.mobilis and CM4 medium for C. thermocellum. A positive control, withpure sugar added, but no feedstock, is used for comparison. During theincubation, a total of five samples is taken over a 12 hour period attime 0, 3, 6, 9, and 12 hours and analyzed for viability (plate countsfor Z. mobilis and direct counts for S. cerevisiae) and ethanolconcentration.

Sugar content of the feedstocks is measured using High PerformanceLiquid Chromatography (HPLC) equipped with either a Shodex® sugar SP0810or Biorad Aminex® HPX-87P column. Each of the feedstocks (approx. 5 g)is mixed with reverse osmosis (RO) water for 1 hour. The liquid portionof the mixture is removed and analyzed for glucose, galactose, xylose,mannose, arabinose, and cellobiose content. The analysis is performedaccording to National Bioenergy Center protocol Determination ofStructural Carbohydrates and Lignin in Biomass.

II. Cellulase Compatibility

Feedstocks are tested, in duplicate, with commercially availableAccellerase® 1000 enzyme complex, which contains a complex of enzymesthat reduces lignocellulosic biomass into fermentable sugars, includingtwo different cellulase preparations, Trichoderma reesei and Aspergillusnidulans, at the recommended temperature and concentration in anErlenmeyer flask. The flasks are incubated with moderate shaking ataround 200 rpm for 12 hours. During that time, samples are taken everythree hours at time 0, 3, 6, 9, and 12 hours to determine theconcentration of reducing sugars (Hope and Dean, Biotech J., 1974,144:403) in the liquid portion of the flasks.

Example 20 Alcohol Production Using Irradiation-Sonication Pretreatment

The optimum size for biomass conversion plants is affected by factorsincluding economies of scale and the type and availability of biomassused as feedstock. Increasing plant size tends to increase economies ofscale associated with plant processes. However, increasing plant sizealso tends to increase the costs (e.g., transportation costs) per unitof biomass feedstock. Studies analyzing these factors suggest that theappropriate size for biomass conversion plants can range from 2000 to10,000 dried tons of biomass feedstock per day. The plant describedbelow is sized to process 2000 tons of dry biomass feedstock per day.

FIG. 39 shows a process schematic of a biomass conversion systemconfigured to process switchgrass. The feed preparation subsystemprocesses raw biomass feedstock to remove foreign objects and provideconsistently sized particles for further processing. The pretreatmentsubsystem changes the molecular structure (e.g., reduces the averagemolecular weight and the crystallinity) of the biomass feedstock byirradiating the biomass feedstock, mixing the irradiated the biomassfeedstock with water to form a slurry, and applying ultrasonic energy tothe slurry. The irradiation and sonication convert the cellulosic andlignocellulosic components of the biomass feedstock into fermentablematerials. The primary process subsystem ferments the glucose and otherlow weight sugars present after pretreatment to form alcohols.

Feed Preparation

The selected design feed rate for the plant is 2,000 dry tons per day ofswitchgrass biomass. The design feed is chopped and/or shearedswitchgrass.

Biomass feedstock in the form of bales of switchgrass is received by theplant. In some cases, the switchgrass bales are wrapped with plastic netto ensure they don't break apart when handled, and can also be wrappedin plastic film to protect the bale from weather. The bales are eithersquare or round. The bales are received at the plant from off-sitestorage on large truck trailers. As the trucks are received, they areweighed and unloaded by forklifts. Some bales are sent to on-sitestorage while others are taken directly to the conveyors.

Since switchgrass is only seasonally available, long-term storage isrequired to provide feed to the plant year-round. Long-term storage willlikely consist of 400-500 acres of uncovered piled rows of bales at alocation (or multiple locations) reasonably close to the ethanol plant.On-site short-term storage is provided equivalent to 72 hours ofproduction at an outside storage area. Bales and surrounding access waysas well as the transport conveyors will be on a concrete slab. Aconcrete slab is used because of the volume of traffic required todeliver the large amount of biomass feedstock required. A concrete slabwill minimize the amount of standing water in the storage area, as wellas reduce the biomass feedstock's exposure to dirt. The stored materialprovides a short-term supply for weekends, holidays, and when normaldirect delivery of material into the process is interrupted.

The bales are off-loaded by forklifts and are placed directly onto baletransport conveyors or in the short-term storage area. Bales are alsoreclaimed from short-term storage by forklifts and loaded onto the baletransport conveyors.

Bales travel to one of two bale unwrapping stations. Unwrapped bales arebroken up using a spreader bar and then discharged onto a conveyor,which passes a magnetic separator to remove metal prior to shredding. Atramp iron magnet is provided to catch stray magnetic metal and ascalping screen removes gross oversize and foreign material ahead ofmultiple shredder-shearer trains, which reduce the biomass feedstock tothe proper size for pretreatment. The shredder-shearer trains includeshredders and rotary knife cutters. The shredders reduce the size of theraw biomass feedstock and feed the resulting material to the rotaryknife cutters. The rotary knife cutters concurrently shear the biomassfeedstock and screen the resulting material. Finally, the biomassfeedstock is conveyed to the pretreatment subsystem.

Three storage silos are provided to limit overall system downtime due torequired maintenance on and/or breakdowns of feed preparation subsystemequipment. Each silo can hold approximately 55,000 cubic feet of biomassfeedstock (˜3 hours of plant operation).

Pretreatment

A conveyor belt carries the biomass feedstock from the feed preparationsubsystem 110 to the pretreatment subsystem 114. As shown in FIG. 40, inthe pretreatment subsystem 114, the biomass feedstock is irradiatedusing electron beam emitters, mixed with water to form a slurry, andsubjected to the application of ultrasonic energy. As discussed above,irradiation of the biomass feedstock changes the molecular structure(e.g., reduces the recalcitrance, the average molecular weight, and thecrystallinity) of the biomass feedstock. Mixing the irradiated biomassfeedstock into a slurry and applying ultrasonic energy to the slurryfurther changes the molecular structure of the biomass feedstock.Application of the radiation and sonication in sequence can havesynergistic effects in that the combination of techniques appears toachieve greater changes to the molecular structure (e.g., reduces therecalcitrance, the average molecular weight, and the crystallinity) thaneither technique can efficiently achieve on its own. Without wishing tobe bound by theory, in addition to reducing the polymerization of thebiomass feedstock by breaking intramolecular bonds between segments ofcellulosic and lignocellulosic components of the biomass feedstock, theirradiation can make the overall physical structure of the biomassfeedstock more brittle. After the brittle biomass feedstock is mixedinto a slurry, the application of ultrasonic energy further changes themolecular structure (e.g., reduces the average molecular weight and thecrystallinity) and also can reduce the size of biomass feedstockparticles.

Electron Beam Irradiation

The conveyor belt 491 carrying the biomass feedstock into thepretreatment subsystem distributes the biomass feedstock into multiplefeed streams (e.g., 50 feed streams) each leading to separate electronbeam emitters 492. In this embodiment, the biomass feedstock isirradiated while it is dry. Each feed stream is carried on a separateconveyor belt to an associated electron beam emitter. Each irradiationfeed conveyor belt can be approximately one meter wide. Before reachingthe electron beam emitter, a localized vibration is induced in eachconveyor belt to evenly distribute the dry biomass feedstock over thecross-sectional width of the conveyor belt.

Electron beam emitter 492 (e.g., electron beam irradiation devicescommercially available from Titan Corporation, San Diego, Calif.) areconfigured to apply a 100 kilo-Gray dose of electrons applied at a powerof 300 kW. The electron beam emitters are scanning beam devices with asweep width of 1 meter to correspond to the width of the conveyor belt.In some embodiments, electron beam emitters with large, fixed beamwidths are used. Factors including belt/beam width, desired dose,biomass feedstock density, and power applied govern the number ofelectron beam emitters required for the plant to process 2,000 tons perday of dry feed.

Sonication

The irradiated biomass feedstock is mixed with water to form a slurrybefore ultrasonic energy is applied. There can be a separate sonicationsystem associated with each electron beam feed stream or severalelectron beam streams can be aggregated as feed for a single sonicationsystem.

In each sonication system, the irradiated biomass feedstock is fed intoa reservoir 1214 through a first intake 1232 and water is fed into thereservoir 1214 through second intake 1234. Appropriate valves (manual orautomated) control the flow of biomass feedstock and the flow of waterto produce a desired ratio of biomass feedstock to water (e.g., 10%cellulosic material, weight by volume). Each reservoir 1214 includes amixer 1240 to agitate the contents of volume 1236 and disperse biomassfeedstock throughout the water.

In each sonication system, the slurry is pumped (e.g., using a recessedimpeller vortex pump 1218) from reservoir 1214 to and through a flowcell 1224 including an ultrasonic transducer 1226. In some embodiments,pump 1218 is configured to agitate the slurry 1216 such that the mixtureof biomass feedstock and water is substantially uniform at inlet 1220 ofthe flow cell 1224. For example, the pump 1218 can agitate the slurry1216 to create a turbulent flow that persists throughout the pipingbetween the first pump and inlet 1220 of flow cell 1224.

Within the flow cell 1224, ultrasonic transducer 1226 transmitsultrasonic energy into slurry 1216 as the slurry flows through flow cell1224. Ultrasonic transducer 1226 converts electrical energy into highfrequency mechanical energy (e.g., ultrasonic energy), which is thendelivered to the slurry through booster 48. Ultrasonic transducers arecommercially available (e.g., from Hielscher USA, Inc. of Ringwood,N.J.) that are capable of delivering a continuous power of 16 kilowatts.

The ultrasonic energy traveling through booster 1248 in reactor volume1244 creates a series of compressions and rarefactions in process stream1216 with an intensity sufficient to create cavitation in process stream1216. Cavitation disaggregates components of the biomass feedstockincluding, for example, cellulosic and lignocellulosic materialdispersed in process stream 1216 (e.g., slurry). Cavitation alsoproduces free radicals in the water of process stream 1216 (e.g.,slurry). These free radicals act to further break down the cellulosicmaterial in process stream 1216. In general, about 250 MJ/m³ ofultrasonic energy is applied to process stream 1216 containing fragmentsof poplar chips. Other levels of ultrasonic energy (between about 5 andabout 4000 MJ/m³, e.g., 10, 25, 50, 100, 250, 500, 750, 1000, 2000, or3000) can be applied to other biomass feedstocks After exposure toultrasonic energy in reactor volume 1244, process stream 1216 exits flowcell 24 through outlet 1222.

Flow cell 1224 also includes a heat exchanger 1246 in thermalcommunication with at least a portion of reactor volume 1244. Coolingfluid 1248 (e.g., water) flows into heat exchanger 1246 and absorbs heatgenerated when process stream 1216 (e.g., slurry) is sonicated inreactor volume 1244. In some embodiments, the flow of cooling fluid 1248into heat exchanger 1246 is controlled to maintain an approximatelyconstant temperature in reactor volume 1244. In addition or in thealternative, the temperature of cooling fluid 1248 flowing into heatexchanger 1246 is controlled to maintain an approximately constanttemperature in reactor volume 1244.

The outlet 1242 of flow cell 1224 is arranged near the bottom ofreservoir 1214 to induce a gravity feed of process stream 1216 (e.g.,slurry) out of reservoir 1214 towards the inlet of a second pump 1230which pumps process stream 1216 (e.g., slurry) towards the primaryprocess subsystem.

Sonication systems can include a single flow path (as described above)or multiple parallel flow paths each with an associated individualsonication units. Multiple sonication units can also be arranged toseries to increase the amount of sonic energy applied to the slurry.

Primary Processes

A vacuum rotary drum type filter removes solids from the slurry beforefermentation. Liquid from the filter is pumped cooled prior to enteringthe fermentors. Filtered solids are passed to passed to thepost-processing subsystem for further processing.

The fermentation tanks are large, low pressure, stainless steel vesselswith conical bottoms and slow speed agitators. Multiple first stagefermentation tanks can be arranged in series. The temperature in thefirst stage fermentation tanks is controlled to 30 degrees centigradeusing external heat exchangers. Yeast is added to the first stagefermentation tank at the head of each series of tanks and carriesthrough to the other tanks in the series.

Second stage fermentation consists of two continuous fermentors inseries. Both fermentors are continuously agitated with slow speedmechanical mixers. Temperature is controlled with chilled water inexternal exchangers with continuous recirculation. Recirculation pumpsare of the progressive cavity type because of the high solidsconcentration.

Off gas from the fermentation tanks and fermentors is combined andwashed in a counter-current water column before being vented to theatmosphere. The off gas is washed to recover ethanol rather than for airemissions control.

Post-Processing

Distillation

Distillation and molecular sieve adsorption are used to recover ethanolfrom the raw fermentation beer and produce 99.5% ethanol. Distillationis accomplished in two columns—the first, called the beer column,removes the dissolved CO₂ and most of the water, and the secondconcentrates the ethanol to a near azeotropic composition.

All the water from the nearly azeotropic mixture is removed by vaporphase molecular sieve adsorption. Regeneration of the adsorption columnsrequires that an ethanol water mixture be recycled to distillation forrecovery.

Fermentation vents (containing mostly CO₂, but also some ethanol) aswell as the beer column vent are scrubbed in a water scrubber,recovering nearly all of the ethanol. The scrubber effluent is fed tothe first distillation column along with the fermentation beer.

The bottoms from the first distillation contain all the unconvertedinsoluble and dissolved solids. The insoluble solids are dewatered by apressure filter and sent to a combustor. The liquid from the pressurefilter that is not recycled is concentrated in a multiple effectevaporator using waste heat from the distillation. The concentratedsyrup from the evaporator is mixed with the solids being sent to thecombustor, and the evaporated condensate is used as relatively cleanrecycle water to the process.

Because the amount of stillage water that can be recycled is limited, anevaporator is included in the process. The total amount of the waterfrom the pressure filter that is directly recycled is set at 25%.Organic salts like ammonium acetate or lactate, steep liquor componentsnot utilized by the organism, or inorganic compounds in the biomass endup in this stream. Recycling too much of this material can result inlevels of ionic strength and osmotic pressures that can be detrimentalto the fermenting organism's efficiency. For the water that is notrecycled, the evaporator concentrates the dissolved solids into a syrupthat can be sent to the combustor, minimizing the load to wastewatertreatment.

Wastewater Treatment

The wastewater treatment section treats process water for reuse toreduce plant makeup water requirements. Wastewater is initially screenedto remove large particles, which are collected in a hopper and sent to alandfill. Screening is followed by anaerobic digestion and aerobicdigestion to digest organic matter in the stream. Anaerobic digestionproduces a biogas stream that is rich in methane that is fed to thecombustor. Aerobic digestion produces a relatively clean water streamfor reuse in the process as well as a sludge that is primarily composedof cell mass. The sludge is also burned in the combustor. Thisscreening/anaerobic digestion/aerobic digestion scheme is standardwithin the current ethanol industry and facilities in the 1-5 milliongallons per day range can be obtained as “off-the-shelf” units fromvendors.

Combustor, Boiler, and Turbo-Generator

The purpose of the combustor, boiler, and turbo-generator subsystem isto burn various by-product streams for steam and electricity generation.For example, some lignin, cellulose, and hemicellulose remainsunconverted through the pretreatment and primary processes. The majorityof wastewater from the process is concentrated to a syrup high insoluble solids. Anaerobic digestion of the remaining wastewater producesa biogas high in methane. Aerobic digestion produces a small amount ofwaste biomass (sludge). Burning these by-product streams to generatesteam and electricity allows the plant to be self sufficient in energy,reduces solid waste disposal costs, and generates additional revenuethrough sales of excess electricity.

Three primary fuel streams (post-distillate solids, biogas, andevaporator syrup) are fed to a circulating fluidized bed combustor. Thesmall amount of waste biomass (sludge) from wastewater treatment is alsosent to the combustor. A fan moves air into the combustion chamber.Treated water enters the heat exchanger circuit in the combustor and isevaporated and superheated to 510° C. (950° F.) and 86 atm (1265 psia)steam. Flue gas from the combustor preheats the entering combustion airthen enters a baghouse to remove particulates, which are landfilled. Thegas is exhausted through a stack.

A multistage turbine and generator are used to generate electricity.Steam is extracted from the turbine at three different conditions forinjection into the pretreatment reactor and heat exchange indistillation and evaporation. The remaining steam is condensed withcooling water and returned to the boiler feedwater system along withcondensate from the various heat exchangers in the process. Treated wellwater is used as makeup to replace steam used in direct injection.

Example 21 Preparation of Animal Feed from Switchrass

A 1500 pound skid of switchgrass is purchased from a farm andtransported to the processing site. The material is fed into a 3 hpFlinch Baugh shredder at a rate of approximately 15 to 20 pounds perhour. The shredder is equipped with two 12 inch rotary blades, two fixedblades and a 0.30 inch discharge screen. The gap between the rotary andfixed blades is adjusted to 0.10 inch. The output from the shredderresembles confetti having a width of between 0.1 inch and 0.5 inch, alength of between 0.25 inch and 1 inch, and a thickness equivalent tothat of the starting material. The confetti-like material is fed to aMunson rotary knife cutter, Model SC30. The discharge screen has ⅛ inchopenings. The gap between the rotary and fixed blades is set toapproximately 0.020 inch. The rotary knife cutter shears theconfetti-like pieces, releasing a fibrous material at a rate of aboutone pound per hour. An average length of the fibers is 1.063 mm and anaverage width of the fibers is 0.0245 mm, giving an average L/D of 43:1.

These processed samples are densified to form pellets suitable forconsumption by cows and other livestock. Pellets are distributed tofarms and are stored in a storage silo. Required amounts of pellets arefed per cow per day.

Example 22 Preparation of Animal Feed from Switchrass

A 1500 pound skid of switchgrass is purchased from a farm andtransported to the processing site. The material is fed into a 3 hpFlinch Baugh shredder at a rate of approximately 15 to 20 pounds perhour. The shredder is equipped with two 12 inch rotary blades, two fixedblades and a 0.30 inch discharge screen. The gap between the rotary andfixed blades is adjusted to 0.10 inch. The output from the shredderresembles confetti having a width of between 0.1 inch and 0.5 inch, alength of between 0.25 inch and 1 inch, and a thickness equivalent tothat of the starting material. The confetti-like material is fed to aMunson rotary knife cutter, Model SC30. The discharge screen has ⅛ inchopenings. The gap between the rotary and fixed blades is set toapproximately 0.020 inch. The rotary knife cutter shears theconfetti-like pieces, releasing a fibrous material at a rate of aboutone pound per hour. An average length of the fibers is 1.063 mm and anaverage width of the fibers is 0.0245 mm, giving an average L/D of 43:1.

Samples are treated with an electron beam using a vaulted Rhodotron®TT200 continuous wave accelerator delivering 5 MeV electrons at 80 kW ofoutput power. Table 10 describes the parameters used. Table 11 reportsthe nominal dose used.

TABLE 10 Rhodotron ® TT 200 Parameters Beam Beam Produced: Acceleratedelectrons Beam energy: Nominal (fixed): 10 MeV (+0 keV-250 keV Energydispersion at 10 Full width half maximum (FWHM) 300 keV Mev: Beam powerat 10 MeV: Guaranteed Operating Range 1 to 80 kW Power ConsumptionStand-by condition <15 kW (vacuum and cooling ON): At 50 kW beam power:<210 kW At 80 kW beam power: <260 kW RF System Frequency: 107.5 ± 1 MHzTetrode type: Thomson TH781 Scanning Horn Nominal Scanning Length 120 cm(measured at 25-35 cm from window): Scanning Range: From 30% to 100% ofNominal Scanning Length Nominal Scanning 100 Hz ± 5% Frequency (at max.scanning length): Scanning Uniformity ±5% (across 90% of NominalScanning Length)

TABLE 11 Dosages Delivered to Samples Total Dosage (MRad) 1 3 5 7 10 1520 30 50 70 100

These processed samples are densified to form pellets suitable forconsumption by cows and other livestock. Pellets are distributed tofarms and are stored in a storage silo. Pellets are fed to cows andother livestock.

Example 23 Preparation of Animal Feed from Alfalfa

A 1500 pound skid of alfalfa is fed into a 3 hp Flinch Baugh shredder ata rate of approximately 15 to 20 pounds per hour. The shredder isequipped with two 12 inch rotary blades, two fixed blades and a 0.30inch discharge screen. The gap between the rotary and fixed blades isadjusted to 0.10 inch. The output from the shredder resembles confettihaving a width of between 0.1 inch and 0.5 inch, a length of between0.25 inch and 1 inch, and a thickness equivalent to that of the startingmaterial. The confetti-like material is fed to a Munson rotary knifecutter, Model SC30. The discharge screen has ⅛ inch openings. The gapbetween the rotary and fixed blades is set to approximately 0.020 inch.The rotary knife cutter shears the confetti-like pieces, releasing afibrous material at a rate of about one pound per hour. An averagelength of the fibers is 1.063 mm and an average width of the fibers is0.0245 mm, giving an average L/D of 43:1.

These processed samples are densified to form pellets suitable forconsumption by cows and other livestock. Pellets are distributed tofarms and are stored in a storage silo. These pellets are fed to cowsand other livestock.

Example 24 Preparation of Animal Feed from Alfalfa

A 1500 pound skid of alfalfa is fed into a 3 hp Flinch Baugh shredder ata rate of approximately 15 to 20 pounds per hour. The shredder isequipped with two 12 inch rotary blades, two fixed blades and a 0.30inch discharge screen. The gap between the rotary and fixed blades isadjusted to 0.10 inch. The output from the shredder resembles confettihaving a width of between 0.1 inch and 0.5 inch, a length of between0.25 inch and 1 inch, and a thickness equivalent to that of the startingmaterial. The confetti-like material is fed to a Munson rotary knifecutter, Model SC30. The discharge screen has ⅛ inch openings. The gapbetween the rotary and fixed blades is set to approximately 0.020 inch.The rotary knife cutter shears the confetti-like pieces, releasing afibrous material at a rate of about one pound per hour. An averagelength of the fibers is 1.063 mm and an average width of the fibers is0.0245 mm, giving an average L/D of 43:1.

Samples are treated with an electron beam using a Rhodotron® TT200continuous wave accelerator delivering 5 MeV electrons at 80 kW ofoutput power. Table 10 describes the parameters used. Table 11 reportsthe nominal dose used.

These processed samples are densified to form pellets suitable forconsumption by cows and other livestock. Pellets are distributed tofarms and are stored in a storage silo. Pellets are fed to cows andother livestock.

Example 25 Preparation of Animal Feed from Paper

A 1500 pound skid of paper is folded flat, and fed into a 3 hp FlinchBaugh shredder at a rate of approximately 15 to 20 pounds per hour. Theshredder is equipped with two 12 inch rotary blades, two fixed bladesand a 0.30 inch discharge screen. The gap between the rotary and fixedblades is adjusted to 0.10 inch. The output from the shredder resemblesconfetti having a width of between 0.1 inch and 0.5 inch, a length ofbetween 0.25 inch and 1 inch, and a thickness equivalent to that of thestarting material. The confetti-like material is fed to a Munson rotaryknife cutter, Model SC30. The discharge screen has ⅛ inch openings. Thegap between the rotary and fixed blades is set to approximately 0.020inch. The rotary knife cutter shears the confetti-like pieces, releasinga fibrous material at a rate of about one pound per hour. An averagelength of the fibers is 1.063 mm and an average width of the fibers is0.0245 mm, giving an average L/D of 43:1.

These processed samples are densified to form pellets suitable forconsumption by cows and other livestock. Pellets are distributed tofarms and are stored in a storage silo. These pellets are fed to cowsand other livestock.

Example 26 Preparation of Animal Feed from Paper

A 1500 pound skid of paper is fed into a 3 hp Flinch Baugh shredder at arate of approximately 15 to 20 pounds per hour. The shredder is equippedwith two 12 inch rotary blades, two fixed blades and a 0.30 inchdischarge screen. The gap between the rotary and fixed blades isadjusted to 0.10 inch. The output from the shredder resembles confettihaving a width of between 0.1 inch and 0.5 inch, a length of between0.25 inch and 1 inch, and a thickness equivalent to that of the startingmaterial. The confetti-like material is fed to a Munson rotary knifecutter, Model SC30. The discharge screen has ⅛ inch openings. The gapbetween the rotary and fixed blades is set to approximately 0.020 inch.The rotary knife cutter shears the confetti-like pieces, releasing afibrous material at a rate of about one pound per hour. An averagelength of the fibers is 1.063 mm and an average width of the fibers is0.0245 mm, giving an average L/D of 43:1.

Samples are treated with an electron beam using a Rhodotron® TT200continuous wave accelerator delivering 5 MeV electrons at 80 kW ofoutput power. Table 10 describes the parameters used. Table 11 reportsthe nominal dose used.

These processed samples are densified to form pellets suitable forconsumption by cows and other livestock. Pellets are distributed tofarms and are stored in a storage silo. Pellets are fed to cows andother livestock.

Example 27 Preparation of Animal Feed from Grass

A 1500 pound gaylord of grass is fed into a 3 hp Flinch Baugh shredderat a rate of approximately 15 to 20 pounds per hour. The shredder isequipped with two 12 inch rotary blades, two fixed blades and a 0.30inch discharge screen. The gap between the rotary and fixed blades isadjusted to 0.10 inch. The output from the shredder resembles confettihaving a width of between 0.1 inch and 0.5 inch, a length of between0.25 inch and 1 inch, and a thickness equivalent to that of the startingmaterial. The confetti-like material is fed to a Munson rotary knifecutter, Model SC30. The discharge screen has ⅛ inch openings. The gapbetween the rotary and fixed blades is set to approximately 0.020 inch.The rotary knife cutter shears the confetti-like pieces, releasing afibrous material at a rate of about one pound per hour. An averagelength of the fibers is 1.063 mm and an average width of the fibers is0.0245 mm, giving an average L/D of 43:1.

Processed samples are densified to form pellets suitable for consumptionby cows and other livestock. Pellets are distributed to farms and arestored in a storage silo. These pellets are fed to cows and otherlivestock.

Example 28 Preparation of Animal Feed from Grass

A 1500 pound skid of grass is fed into a 3 hp Flinch Baugh shredder at arate of approximately 15 to 20 pounds per hour. The shredder is equippedwith two 12 inch rotary blades, two fixed blades and a 0.30 inchdischarge screen. The gap between the rotary and fixed blades isadjusted to 0.10 inch. The output from the shredder resembles confettihaving a width of between 0.1 inch and 0.5 inch, a length of between0.25 inch and 1 inch, and a thickness equivalent to that of the startingmaterial. The confetti-like material is fed to a Munson rotary knifecutter, Model SC30. The discharge screen has ⅛ inch openings. The gapbetween the rotary and fixed blades is set to approximately 0.020 inch.The rotary knife cutter shears the confetti-like pieces, releasing afibrous material at a rate of about one pound per hour. An averagelength of the fibers is 1.063 mm and an average width of the fibers is0.0245 mm, giving an average L/D of 43:1.

Samples are treated with an electron beam using a Rhodotron® TT200continuous wave accelerator delivering 5 MeV electrons at 80 kW ofoutput power. Table 10 describes the parameters used. Table 11 reportsthe nominal dose used.

These processed samples are densified to form pellets suitable forconsumption by cows and other livestock. Pellets are distributed tofarms and are stored in a storage silo. Pellets are fed to cows andother livestock.

Example 29 Preparation of Animal Feed from Wheatstraw

A 1500 pound skid of wheatstraw is fed into a 3 hp Flinch Baugh shredderat a rate of approximately 15 to 20 pounds per hour. The shredder isequipped with two 12 inch rotary blades, two fixed blades and a 0.30inch discharge screen. The gap between the rotary and fixed blades isadjusted to 0.10 inch. The output from the shredder resembles confettihaving a width of between 0.1 inch and 0.5 inch, a length of between0.25 inch and 1 inch, and a thickness equivalent to that of the startingmaterial. The confetti-like material is fed to a Munson rotary knifecutter, Model SC30. The discharge screen has ⅛ inch openings. The gapbetween the rotary and fixed blades is set to approximately 0.020 inch.The rotary knife cutter shears the confetti-like pieces, releasing afibrous material at a rate of about one pound per hour. An averagelength of the fibers is 1.063 mm and an average width of the fibers is0.0245 mm, giving an average L/D of 43:1.

Processed samples are densified to form pellets suitable for consumptionby cows and other livestock. Pellets are distributed to farms and arestored in a storage silo. These pellets are fed to cows and otherlivestock.

Example 30 Preparation of Animal Feed from Wheatstraw

A 1500 pound skid of wheatstraw is fed into a 3 hp Flinch Baugh shredderat a rate of approximately 15 to 20 pounds per hour. The shredder isequipped with two 12 inch rotary blades, two fixed blades and a 0.30inch discharge screen. The gap between the rotary and fixed blades isadjusted to 0.10 inch. The output from the shredder resembles confettihaving a width of between 0.1 inch and 0.5 inch, a length of between0.25 inch and 1 inch, and a thickness equivalent to that of the startingmaterial. The confetti-like material is fed to a Munson rotary knifecutter, Model SC30. The discharge screen has ⅛ inch openings. The gapbetween the rotary and fixed blades is set to approximately 0.020 inch.The rotary knife cutter shears the confetti-like pieces, releasing afibrous material at a rate of about one pound per hour. An averagelength of the fibers is 1.063 mm and an average width of the fibers is0.0245 mm, giving an average L/D of 43:1.

Samples are treated with an electron beam using a Rhodotron® TT200continuous wave accelerator delivering 5 MeV electrons at 80 kW ofoutput power. Table 10 describes the parameters used. Table 11 reportsthe nominal dose used.

These processed samples are densified to form pellets suitable forconsumption by cows and other livestock. Pellets are distributed tofarms and are stored in a storage silo. Pellets are fed to cows andother livestock.

Example 31 Preparation of Animal Feed from Biomass

1500 pound skids of switchgrass, alfalfa, paper, grass, and wheatstraware fed separately into a 3 hp Flinch Baugh shredder at a rate ofapproximately 15 to 20 pounds per hour. The shredder is equipped withtwo 12 inch rotary blades, two fixed blades and a 0.30 inch dischargescreen. The gap between the rotary and fixed blades is adjusted to 0.10inch. The output from the shredder resembles confetti having a width ofbetween 0.1 inch and 0.5 inch, a length of between 0.25 inch and 1 inch,and a thickness equivalent to that of the starting material. Theconfetti-like material is fed to a Munson rotary knife cutter, ModelSC30. The discharge screen has ⅛ inch openings. The gap between therotary and fixed blades is set to approximately 0.020 inch. The rotaryknife cutter shears the confetti-like pieces, releasing a fibrousmaterial at a rate of about one pound per hour. An average length of thefibers is 1.063 mm and an average width of the fibers is 0.0245 mm,giving an average L/D of 43:1.

Processed samples are combined and densified to form pellets suitablefor consumption by cows and other livestock. Pellets are distributed tofarms and are stored in a storage silo. These pellets are fed to cowsand other livestock.

Example 32 Preparation of Animal Feed from Biomass

1500 pound skids of switchgrass, alfalfa, paper, grass, and wheatstraware fed separately into a 3 hp Flinch Baugh shredder at a rate ofapproximately 15 to 20 pounds per hour. The shredder is equipped withtwo 12 inch rotary blades, two fixed blades and a 0.30 inch dischargescreen. The gap between the rotary and fixed blades is adjusted to 0.10inch. The output from the shredder resembles confetti having a width ofbetween 0.1 inch and 0.5 inch, a length of between 0.25 inch and 1 inch,and a thickness equivalent to that of the starting material. Theconfetti-like material is fed to a Munson rotary knife cutter, ModelSC30. The discharge screen has ⅛ inch openings. The gap between therotary and fixed blades is set to approximately 0.020 inch. The rotaryknife cutter shears the confetti-like pieces, releasing a fibrousmaterial at a rate of about one pound per hour. An average length of thefibers is 1.063 mm and an average width of the fibers is 0.0245 mm,giving an average L/D of 43:1.

Samples are treated with an electron beam using a Rhodotron® TT200continuous wave accelerator delivering 5 MeV electrons at 80 kW ofoutput power. Table 10 describes the parameters used. Table 11 reportsthe nominal dose used.

Processed samples are combined and densified to form pellets suitablefor consumption by cows and other livestock. Pellets are distributed tofarms and are stored in a storage silo. These pellets are fed to cowsand other livestock.

Example 33 Preparation of Animal Feed from Biomass

1500 pound skids of switchgrass, alfalfa, paper, grass, and wheatstraware mixed and fed into a 3 hp Flinch Baugh shredder at a rate ofapproximately 15 to 20 pounds per hour. The shredder is equipped withtwo 12 inch rotary blades, two fixed blades and a 0.30 inch dischargescreen. The gap between the rotary and fixed blades is adjusted to 0.10inch. The output from the shredder resembles confetti having a width ofbetween 0.1 inch and 0.5 inch, a length of between 0.25 inch and 1 inch,and a thickness equivalent to that of the starting material. Theconfetti-like material is fed to a Munson rotary knife cutter, ModelSC30. The discharge screen has ⅛ inch openings. The gap between therotary and fixed blades is set to approximately 0.020 inch. The rotaryknife cutter shears the confetti-like pieces, releasing a fibrousmaterial at a rate of about one pound per hour. An average length of thefibers is 1.063 mm and an average width of the fibers is 0.0245 mm,giving an average L/D of 43:1.

Processed samples are densified to form pellets suitable for consumptionby cows and other livestock. Pellets are distributed to farms and arestored in a storage silo. These pellets are fed to cows and otherlivestock.

Example 34 Preparation of Animal Feed from Biomass

1500 pound skids of switchgrass, alfalfa, paper, grass, and wheatstraware mixed and fed into a 3 hp Flinch Baugh shredder at a rate ofapproximately 15 to 20 pounds per hour. The shredder is equipped withtwo 12 inch rotary blades, two fixed blades and a 0.30 inch dischargescreen. The gap between the rotary and fixed blades is adjusted to 0.10inch. The output from the shredder resembles confetti having a width ofbetween 0.1 inch and 0.5 inch, a length of between 0.25 inch and 1 inch,and a thickness equivalent to that of the starting material. Theconfetti-like material is fed to a Munson rotary knife cutter, ModelSC30. The discharge screen has ⅛ inch openings. The gap between therotary and fixed blades is set to approximately 0.020 inch. The rotaryknife cutter shears the confetti-like pieces, releasing a fibrousmaterial at a rate of about one pound per hour. An average length of thefibers is 1.063 mm and an average width of the fibers is 0.0245 mm,giving an average L/D of 43:1.

Samples are treated with an electron beam using a Rhodotron® TT200continuous wave accelerator delivering 5 MeV electrons at 80 kW ofoutput power. Table 10 describes the parameters used. Table 11 reportsthe nominal dose used.

Processed samples are densified to form pellets suitable for consumptionby cows and other livestock. Pellets are distributed to farms and arestored in a storage silo. These pellets are fed to cows and otherlivestock.

Example 35 Preparation of Animal Feed from Biomass

1500 pound skids of switchgrass, alfalfa, paper, grass, and wheatstraware mixed and fed into a 3 hp Flinch Baugh shredder at a rate ofapproximately 15 to 20 pounds per hour. The shredder is equipped withtwo 12 inch rotary blades, two fixed blades and a 0.30 inch dischargescreen. The gap between the rotary and fixed blades is adjusted to 0.10inch. The output from the shredder resembles confetti having a width ofbetween 0.1 inch and 0.5 inch, a length of between 0.25 inch and 1 inch,and a thickness equivalent to that of the starting material. Theconfetti-like material is fed to a Munson rotary knife cutter, ModelSC30. The discharge screen has ⅛ inch openings. The gap between therotary and fixed blades is set to approximately 0.020 inch. The rotaryknife cutter shears the confetti-like pieces, releasing a fibrousmaterial at a rate of about one pound per hour. An average length of thefibers is 1.063 mm and an average width of the fibers is 0.0245 mm,giving an average L/D of 43:1.

Processed samples are combined with dried distillers grains (DDG) toproduce a mixture suitable for consumption by cows and other livestock.These mixtures are distributed to farms and are stored in a storagesilo. These pellets are fed to cows and other livestock.

Example 36 Preparation of Animal Feed from Biomass

1500 pound skids of switchgrass, alfalfa, paper, grass, and wheatstraware mixed and fed into a 3 hp Flinch Baugh shredder at a rate ofapproximately 15 to 20 pounds per hour. The shredder is equipped withtwo 12 inch rotary blades, two fixed blades and a 0.30 inch dischargescreen. The gap between the rotary and fixed blades is adjusted to 0.10inch. The output from the shredder resembles confetti having a width ofbetween 0.1 inch and 0.5 inch, a length of between 0.25 inch and 1 inch,and a thickness equivalent to that of the starting material. Theconfetti-like material is fed to a Munson rotary knife cutter, ModelSC30. The discharge screen has ⅛ inch openings. The gap between therotary and fixed blades is set to approximately 0.020 inch. The rotaryknife cutter shears the confetti-like pieces, releasing a fibrousmaterial at a rate of about one pound per hour. An average length of thefibers is 1.063 mm and an average width of the fibers is 0.0245 mm,giving an average L/D of 43:1.

Samples are treated with an electron beam using a Rhodotron® TT200continuous wave accelerator delivering 5 MeV electrons at 80 kW ofoutput power. Table 10 describes the parameters used. Table 11 reportsthe nominal dose used.

Processed samples are combined with dried distillers grains (DDG) toproduce a mixture suitable for consumption by cows and other livestock.These mixtures are distributed to farms and are stored in a storagesilo. These pellets are fed to cows and other livestock.

Example 37 Self Sufficient Farming

A farmer collects a crop of switchgrass and sends it for processing to aprocessing plant. The switchgrass is processed as described in Example21. The processed material is supplied to the farmer in the form of apellet that is fed to the farmer's cows and other livestock.

Example 38 Self Sufficient Farming

A farmer collects a crop of switchgrass and sends it for processing to aprocessing plant. The switchgrass is processed as described in Example22. The processed material is supplied to the farmer in the form of apellet that is fed to the farmer's cows and other livestock.

Example 39 Self Sufficient Farming

A farmer collects a crop of switchgrass and processes the material usingequipment located on site at the farm. The switchgrass is processed asdescribed in Example 21. The processed material is fed to the farmer'scows and other livestock.

Example 40 Self Sufficient Farming

A farmer collects a crop of switchgrass and processes the material usingequipment located on site at the farm. The switchgrass is processed asdescribed in Example 22. The processed material is fed to the farmer'scows and other livestock.

Example 41 Shake Flask Fermentation Studies Using P. stipitis Summary

Shake flask fermentation studies using various enzymes, physicaltreatments, and Pichia stipitis were performed.

Protocol

Experiments were performed under the parameters outlined in Table 13.

TABLE 13 Chemicals and Materials Used for the Shake Flask ExperimentMedia Component Manufacturer Reference # Urea ScholAR 9472706  ChemistryYeast Nitrogen Becton 291940 Base Dickinson Peptone Becton 211677Dickinson Xylose Alfa Aesar A10643 Glucose Sigma G-5400 Yeast ExtractBecton 288620 Dickinson YM Broth Becton 271120 Dickinson Novozyme ® 188Novozymes Sigma #C6105 Celluclast 1,5 FG Novozymes Sigma #C2730 SolkaFloc International 200 NF Fibre Corporation Pluronic F-68 Sigma P1300Accellerase ® 1000 Genencor N/A

Seed Development

A working cell bank of P. stipitis NRRL Y-7124 was prepared from arehydrated lyophilized culture obtained from ARS Culture Collection.Cryovials containing P. stipitis culture in 15% v/v glycerol were storedat −75° C. A portion of the thawed working cell bank material werestreaked onto a Yeast Mold (YM) Broth+20 g/L agar (pH 5.0) and incubatedat 30° C. for 2 days. The plates were held for up to seven days at 4° C.before use.

A 250 mL Erlenmeyer flask containing 100 mL of medium (40 g/L glucose,1.7 g/L yeast nitrogen base, 2.27 g/L urea, 6.56 g/L peptone, 40 g/Lxylose, pH 5.0) were inoculated with one colony and incubated for 24hours at 25° C. and 150 rpm. After 23 hours of growth, a sample wastaken and analyzed for optical density (OD 600 nm in a UVspectrophotometer) and purity (Gram stain). Based on these results, twoseed flasks, each having an optical density (OD) of between 4 and 8 andwith a clean Gram stain, were combined to inoculate the growth flasks.

Exemplary Experiments

Experiments were performed to 1) determine the correct sonifier outputand temperature regulation (below 60° C.) and 2) confirm theconcentration of Celluclast 1,5 FG and Novozyme 188 with and withoutPluronic F-68.

Five hundred milliliters of water were added to a 1 L glass beaker. Thehorn of a Branson Model 450 Sonifier was placed ½ inch into the surfaceof the beaker and set at a maximum constant output for 60 minutes. Thetemperature of the water was measured every 10 minutes for 60 minutes ofsonication.

An experiment was performed to determine if 1) the concentration ofCelluclast 1,5 FG and Novozyme 188 (0.5 mL and 0.1 mL per gram ofbiomass, respectively) was sufficient for the shake flask experimentsand 2) if the addition of Pluronic F68 augmented the hydrolysis ofcellulose. Four 250 mL flasks were prepared with 100 mL of sterile broth(1.7 g/L yeast nitrogen base, 2.27 g/L urea, 6.56 g/L peptone, pH 5.0).Duplicate flasks contained 1% w/v Pluronic F-68. Solka Floc CrystallineCellulose (6 g) was added to the flasks and allowed to soak at roomtemperature for 14 hours. Celluclast 1,5 FG and Novozyme 188 (0.5 mL and0.1 mL per gram of Solka Floc, respectively) were added and each flaskincubated at 50° C. for 24 hours at 100 rpm. Samples were taken prior tothe addition of enzyme and 24 hours post enzyme addition from all fourflasks and analyzed for glucose concentration using the YSI BiochemAnalyzer (YSI, Interscience). One milliliter of Pichia stipitis seedflask contents was added to the four flasks and incubated at 25° C. and125 rpm for 24 hours. Samples were taken from each flask prior toinoculation and after 24 hours incubation and analyzed for ethanolconcentration using the YSI Biochem Analyzer (YSI, Interscience).

Test Flasks

The test flasks were 2.8 L Fernbach flasks holding 900 mL of broth (1.7g/L yeast nitrogen base, 2.27 g/L urea, 6.56 g/L peptone, pH 5.0).Control flasks were 250 mL flasks containing 100 mL of broth (40 g/Lglucose, 1.7 g/L yeast nitrogen base, 2.27 g/L urea, 6.56 g/L peptone,40 g/L xylose, pH 5.0). The exact nature of each flask was decided byXyleco and is described in Table 80 below.

Samples were not sterilized prior to the start of the experiment. Allsamples were added to the flasks and allowed to soak for 15 hours atroom temperature. Some of the samples were sonicated for one hour usinga Branson Model 450 Sonifier equipped with a ½ inch disruptor horn. Theoriginal plan was to split the flask contents into two, and sonicateeach half continuously at the maximum output for the equipment up to 450watts (the allowable output depends on the viscosity of the sample) for1 hour. An output setting of 3 and a Duty cycle of Pulse 90% weresufficient for the mixing of the beaker contents. At an output settingof 3, the meter read between 30 and 40. The output was calculated to be40-60 watts.

Originally, the plan was to mix some samples (see Table 80) for varioustimes using a POLYTRON PT 10/35 laboratory homogenizer (or rotor/stator)at 25,000 rpm for various times. Samples #22 and #23 were split into twobeakers and treated for 30 minutes using the large Kinematica PolytronPT 10/35. The generator (tip) was a PTA 20 with a stator diameter of 20mm. The instrument was operated at a speed of 11,000 rpm. Operationabove 11,000 rpm caused splattering of beaker contents, movement of thebeaker, and over-heating of the equipment. After samples #23 and #24,the Polytron PT 10/35 stopped working, presumably from over-use withquite viscous samples. Therefore, the hand-held Polytron PT1200C wasused. The generator (tip) was a PT-DA 1212 with a stator diameter of 12mm. The instrument could be operated at 25,000 rpm. It was noted by theoperator that a similar degree of mixing was observed with the hand-heldat 25,000 rpm as compared to the larger model at 11,000 rpm. The samplewas periodically mixed by the operator to ensure even mixing. Samples 19through 22 were mixed with the hand-held Polytron PT1200C.

Enzyme pretreatments included: 1) E1=Accellerase® 1000 enzyme complex ata loading density of 0.25 mL per gram of substrate and 2) E2=Celluclast1,5 FG and Novozyme 188 at a loading concentration of 0.5 and 0.1 mL pergram of substrate, respectively. After physical pretreatment (see Table80 below), the appropriate enzyme(s) were added and the flasks held at50° C. and 125 rpm for 20 hours. After 20 hours, the flasks were cooledto room temperature for 1 hour prior to the addition of P. stipitis.

TABLE 14 Summary of Test Treatments Sample Enzyme Concen- TreatmentSample tration Physical (50° C., Test Number Number (g/900 mL) Treatment21 hours) Control (250 mL None — — — flask) performed in duplicate eachweek Week 1 1 SP 35 15 h r.t. soak None 2 XP 35 15 h r.t. soak None 3 SP35 15 h r.t. soak E1 4 SP 35 15 h r.t. soak E2 5 XP 35 15 h r.t. soak E16 XP 35 15 h r.t. soak E2 7 XP-10e 35 15 h r.t. soak E2 8 XP-30e 35 15 hr.t. soak E2 9 XP-50e 35 15 h r.t. soak E2 10 XP-10e 35 15 h r.t. soak,1 E2 hour sonication 11 XP-30e 35 15 h r.t. soak, 1 E2 hour sonication12 XP-50e 35 15 h r.t. soak, 1 E2 hour sonication Week 2 13 XP-10e 35 15h r.t. soak, 10 E2 min sonication 14 XP-30e 35 15 h r.t. soak, 10 E2 minsonication 15 XP-50e 35 15 h r.t. soak, 10 E2 min sonication 16 XP-10e35 15 h r.t. soak, 30 E2 min sonication 17 XP-30e 35 15 h r.t. soak, 30E2 min sonication 18 XP-50e 35 15 h r.t. soak, 30 E2 min sonication 19XP-10e 35 15 h r.t. soak, 10 E2 min rotor/stator 20 XP-30e 35 15 h r.t.soak, 10 E2 min rotor/stator 21 XP-50e 35 15 h r.t. soak, 10 E2 minrotor/stator 22 XP-10e 35 15 h r.t. soak, 30 E2 min rotor/stator 23XP-30e 35 15 h r.t. soak, 30 E2 min rotor/stator 24 XP-50e 35 15 h r.t.soak, 30 E2 min rotor/stator

Analysis

A sample was taken from each flask after physical and/or enzymepretreatment (just prior to the addition of P. stipitis) and analyzedfor glucose concentration using the YSI Biochem Analyzer (YSI,Interscience). Samples were centrifuged at 14,000 rpm for 20 minutes andthe supernatant stored at −20° C. The samples were diluted to between0-25.0 g/L glucose prior to analysis. A glucose standard was analyzedapproximately every 30 samples to ensure the integrity of the membranewas maintained.

A total of five samples were taken from each flask at 0, 12, 24, 48, and72 hours and analyzed for ethanol concentration using the YSI BiochemAnalyzer based on the alcohol dehydrogenase assay (YSI, Interscience).Samples were centrifuged at 14,000 rpm for 20 minutes and thesupernatant stored at −20° C. and diluted to between 0-3.0 g/L ethanolprior to analysis. A standard of 2.0 g/L ethanol was analyzedapproximately every 30 samples to ensure the integrity of the membranewas maintained.

A sample of the seed flask was analyzed in order to determine theinitial cell concentration in the test flasks. In addition one sample at72 hours of incubation was taken from each flask and analyzed for cellconcentration. Appropriately diluted samples were mixed with 0.05%Trypan blue and loaded into a Neubauer haemocytometer. The cells werecounted under 40× magnification.

Results Experiments

The results of a sonifier experiment are presented in Table 81. Therewere no problems with over-heating of the water.

TABLE 15 Sonifier Experiment Time Temperature (° C.) 0 18 10 18 20 19 3019 40 19 50 19 60 19

The results of the experiment to confirm the concentration of Celluclast1,5 FG and Novozyme 188 with and without Pluronic F-68 are presented inTable 82 and 83. A concentration of 60 g/L cellulose (Solka Floc) wasadded to each flask. After 24 hours of incubation, 33.7 to 35.7 g/Lglucose was generated (30.3 to 32.1 g/L cellulose digested).

After 24 hours of incubation with P. stipitis, 23.2-25.7 g/L of glucoseremained in the flasks. This indicates that not all of the glucose wasused within 24 hours of incubation.

There was no evidence of Pluronic F-68 toxicity toward P. stipitis.However, there was no increase in the amount of glucose generated aftera 24 hour enzyme treatment with the addition of Pluronic F-68.

TABLE 16 Glucose Results Glucose Concentration (g/L) Prior After EnzymeAfter to Enzyme Treatment (50° C., P. stipitis Flask Treatment 24 hours,100 rpm for 24 hours Control A 0.28 34.3 23.2 Control B 0.64 35.7 25.3Containing Pluronic A 0.48 34.8 25.6 Containing Pluronic B 0.93 33.725.7

TABLE 17 Ethanol Results Ethanol Concentration (g/L) at times (hours) 0(inoculation, 24 hours of Flask after enzyme treatment) P. stipitisControl A 0.01 7.23 Control B 0.01 5.75 Containing Pluronic A 0.01 7.57Containing Pluronic B 0.00 7.36

During week one of testing, the seed flask had an optical density (600nm) of 9.74 and a cell concentration of 4.21×10⁸ cells/mL. Nine mL ofseed flask material was added to each of the test flasks and 1 mL to thecontrol flasks (1% v/v). Therefore, the starting cell concentration ineach flask was x 4.21×10⁶/mL.

During week two of testing, the seed flask had an optical density (600nm) of 3.02 and a cell concentration of 2.85×10⁸ cells/mL. To accountfor differences in cell counts and OD, 12 mL of seed flask material wasadded to each of the test flasks and 1.5 mL to the control flasks (1.5%v/v). Therefore, the starting cell concentration in each flask was3.80×10⁶/mL.

The ethanol concentration in the flasks is presented in Table 84. Thehighest concentration of ethanol was observed in Flask #6 (Sample XP,Overnight Soak, treatment with E2 at 50° C. for 21 hours). Aconcentration of 19.5 g/L (17.55 g/per flask) was generated from anoriginal 35 grams of substrate in 48 hours. The yield of ethanol (gramsof ethanol/gram of substrate) in flask #6 was 0.50.

TABLE 18 Ethanol Concentration Sample Ethanol Concentration (g/L) atIncubation Time (hours) Number 0 12 24 48 72 Control A 0.249 1.57 9.3113.60 14.20 Control B 0.237 1.04 7.97 11.40 13.90 1 0.247 0.16 0.10 0.110.06 2 0.175 0.12 0.10 0.17 0.29 3 0.284 2.73* 8.88 9.72 10.40 4 0.3980.43 8.02 14.40 12.10 5 0.312 0.31 10.30 11.30 18.80 6 0.399 0.73 7.5519.50* 19.00 7 0.419 0.38 4.73 16.80* 15.40 8 0.370 0.46 0.56 9.86 13.509 0.183 0.47 0.53 12.00 14.10 10 0.216 0.35 6.11 13.80 15.60 11 0.1990.33 0.88 9.02 8.52 12 0.264 0.43 0.41 8.76 13.80 Control A 0.49 0.847.93 13.00 15.00 Control B 0.50 0.93 8.39 13.40 15.00 13 0.86 0.99 8.4210.50 14.20 14 0.95 0.88 3.79 10.90 12.40 15 1.18 0.42 1.12 9.26 12.6016 0.88 0.42 5.41 6.78 12.80 17 0.99 0.45 1.73 10.60 12.00 18 1.17 0.461.12 10.60 12.10 19 0.78 0.50 9.75 12.60 13.40 20 0.94 0.39 2.54 11.1012.20 21 1.28 0.43 1.46 11.50 11.30 22 0.84 1.09 10.00 14.00 10.10 230.96 0.57 6.77 11.10 12.10 24 1.20 0.42 1.91 12.10 13.10 *Samplesanalyzed twice with the same result. Flasks with a concentration ofgreater than 15 g/L ethanol are in BOLD.

The results of the glucose analysis are presented in Table 85. After 21hours of enzyme treatment, the highest concentration of glucose was 19.6g/L (17.6 grams per flask) in flask #6 (Sample XP, Overnight Soak,treatment with E2 at 50° C. for 21 hours). This was also the flask withthe highest ethanol concentration (see Table 84). After 72 hours, verylittle glucose remained in the flasks. No glucose was detected in Flasks1 and 2.

TABLE 19 Glucose Concentration Glucose Concentration (g/L) at SampleIncubation Time (hours) Number 0 72 1 0.0 0.00 2 0.0 0.00 3 7.2 0.02 413.3 0.03 5 15.9 0.05 6 19.6 0.05 7 13.9 0.04 8 15.4 0.06 9 18.3 0.09 1017.1 0.05 11 13.0 0.04 12 17.0 0.08 13 14.4 0.03 14 13.7 0.04 15 16.30.08 16 13.2 0.03 17 13.4 0.04 18 15.8 0.06 19 15.3 0.04 20 14.3 0.04 2115.5 0.06 22 14.7 0.04 23 13.5 0.04 24 16.6 0.07

The results of the direct cell counts are presented in Table 86. Theconcentration of viable cells was higher in the control flasks. Thelowest counts were observed in flasks 1 through 4.

TABLE 20 Cell Counts Sample Number of Cells (×106/mL) after Number 72hours of incubation Control A 38.30 Control B 104.00 1 0.02 2 0.08 30.07 4 0.06 5 0.15 6 1.05 7 1.50 8 1.95 9 1.05 10 3.60 11 1.28 12 0.90Control A 39.80 Control B 30.80 13 0.98 14 0.40 15 0.63 16 0.71 17 1.1518 0.83 19 1.25 20 1.02 21 0.53 22 0.56 23 0.59 24 0.59

Example 42 Production of Bioconversion Products from Biomass

A 150 pound skid of biomass is fed into a 3 hp Flinch Baugh shredder ata rate of approximately 15 to 20 pounds per hour. The shredder isequipped with two 12 inch rotary blades, two fixed blades and a 0.30inch discharge screen. The gap between the rotary and fixed blades isadjusted to 0.10 inch. The output from the shredder resembles confettihaving a width of between 0.1 inch and 0.5 inch, a length of between0.25 inch and 1 inch, and a thickness equivalent to that of the startingmaterial. The confetti-like material is fed to a Munson rotary knifecutter, Model SC30. The discharge screen has ⅛ inch openings. The gapbetween the rotary and fixed blades is set to approximately 0.020 inch.The rotary knife cutter shears the confetti-like pieces, releasing afibrous material at a rate of about one pound per hour. An averagelength of the fibers is 1.063 mm and an average width of the fibers is0.0245 mm, giving an average L/D of 43:1.

Materials are treated with electron beam using a Rhodotron® TT200continuous wave accelerator delivering 5 MeV electrons at 80 kW ofoutput power. Table 10 describes the parameters used. Table 11 reportsthe nominal dose used.

The processed materials are dispensed into New Brunswick Scientificsterilizable bench top sterilizable fermenters00 in the form of a liquidmedium that is formulated to support the growth, expansion, and/oractivity of a microorganism selected for its ability to produce therequired bioconversion product. Varying concentrations of the processedmaterials are added in combination with varying amounts of othersupplementary materials that are routinely necessary for the growth,expansion, and/or activity of the selected microorganism. A nitrogensource is also added to the medium. The concentration or amount of theprocessed materials and each of the supplementary materials (includingthe nitrogen source) are memorialized in the form of a laboratorynotebook or on a computer hard drive.

A starter culture of the selected microorganism is added to each of thevarious culture solutions in the fermentors. Each of the inoculatedculture solutions is incubated at a temperature between about 15° C. andabout 40° C. for 4 to 48 hours under aerobic or an anaerobic conditions.Following culture, microorganisms and cell supernatants are collectedand optionally separated using centrifugation. Samples are then eitherfrozen for storage or are assessed to determine the level ofbioconversion product in the cells or supernatant. Results arememorialized and experiments are repeated until the maximum yield ofbioconversion product is obtained. The culture solution and conditionsused to obtain this maximum yield are scaled up for use in large scalefermentation.

Example 43 Large Scale Production of Bioconversion Products from Biomass

A 1500 pound skid of biomass is fed into a 3 hp Flinch Baugh shredder ata rate of approximately 15 to 20 pounds per hour. The shredder isequipped with two 12 inch rotary blades, two fixed blades and a 0.30inch discharge screen. The gap between the rotary and fixed blades isadjusted to 0.10 inch. The output from the shredder resembles confettihaving a width of between 0.1 inch and 0.5 inch, a length of between0.25 inch and 1 inch, and a thickness equivalent to that of the startingmaterial. The confetti-like material is fed to a Munson rotary knifecutter, Model SC30. The discharge screen has ⅛ inch openings. The gapbetween the rotary and fixed blades is set to approximately 0.020 inch.The rotary knife cutter shears the confetti-like pieces, releasing afibrous material at a rate of about one pound per hour. An averagelength of the fibers is 1.063 mm and an average width of the fibers is0.0245 mm, giving an average L/D of 43:1.

Materials are treated with electron beam using a Rhodotron® TT200continuous wave accelerator delivering 5 MeV electrons at 80 kW ofoutput power. Table 10 describes the parameters used. Table 11 reportsthe nominal dose used.

The processed materials are used in the preparation of the culturesolution determined in Example 42. The selected microorganism andculture solution are combined in a large volume fixed volume fed-batchfermentor and are maintained using the conditions and for the timeperiod determined in Example 42. Concentrated culture solutioncontaining processed materials is added as required to the fermentor. Inaddition, bioconversion product and microorganisms are removed from thefermentor and are processed for storage or use.

Example 44 Large Scale Production of Bioconversion Products from BiomassUsing Animal Waste as a Nitrogen Source

Bioconversion products are produced as described in Example 43 usinganimal waste as a source of nitrogen. Prior to use, animal waste issterilized using filtration or steam and high pressure sterilization.Prior to addition to the culture solution, the sterilized animal wasteis dried.

Example 45 Large Scale Production of Fusarium venenatum (ATCC 20334)from Biomass

Fusarium venenatum is cultured using the process described in Example43. Harvested F. venenatum is combined with rehydrated egg white,onions, textured wheat protein (wheat protein, wheat starch), and canolaoil, and processed for use as a human food.

Other Embodiments

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications can bemade without departing from the spirit and scope of the invention.

In some embodiments, relatively low doses of radiation, optionally,combined with acoustic energy, e.g., ultrasound, are utilized tocross-link, graft, or otherwise increase the molecular weight of anatural or synthetic carbohydrate-containing material, such as any ofthose materials in any form (e.g., fibrous form) described herein, e.g.,sheared or un-sheared cellulosic or lignocellulosic materials, such ascellulose. The cross-linking, grafting, or otherwise increasing themolecular weight of the natural or synthetic carbohydrate-containingmaterial can be performed in a controlled and predetermined manner byselecting the type or types of radiation employed (e.g., e-beam andultraviolet or e-beam and gamma) and/or dose or number of doses ofradiation applied.

For example, a fibrous material that includes a first cellulosic and/orlignocellulosic material having a first molecular weight can beirradiated in a manner to provide a second cellulosic and/orlignocellulosic material having a second molecular weight higher thanthe first molecular weight. For example, if gamma radiation is utilizedas the radiation source, a dose of from about 0.2 Mrad to about 10 Mrad,e.g., from about 0.5 Mrad to about 7.5 Mrad, or from about 2.0 Mrad toabout 5.0 Mrad, can be applied. If e-beam radiation is utilized, asmaller dose can be utilized (relative to gamma radiation), such as adose of from about 0.1 Mrad to about 5 Mrad, e.g., between about 0.2Mrad to about 3 Mrad, or between about 0.25 Mrad and about 2.5 Mrad.

Any of the following additives can added to the fibrous materials,densified fibrous materials or any other materials described herein.Additives, e.g., in the form of a solid, a liquid or a gas, can beadded. Additives include fillers such as calcium carbonate, silica, andtalc; inorganic flame retardants such as alumina trihydrate or magnesiumhydroxide; and organic flame retardants such as chlorinated orbrominated organic compound. Other additives include lignin, fragrances,compatibilizers, processing aids, antioxidants, opacifiers, heatstabilizers, colorants, foaming agents, polymers, e.g., degradablepolymers, photostabilizers, biocides, and antistatic agents, e.g.,stearates or ethoxylated fatty acid amines. Suitable antistaticcompounds include conductive carbon blacks, carbon fibers, metalfillers, cationic compounds, e.g., quaternary ammonium compounds, e.g.,N-(3-chloro-2-hydroxypropyl)-trimethylammonium chloride, alkanolamides,and amines. Representative degradable polymers include polyhydroxyacids, e.g., polylactides, polyglycolides and copolymers of lactic acidand glycolic acid, poly(hydroxybutyric acid), poly(hydroxyvaleric acid),poly[lactide-co-(e-caprolactone)], poly[glycolide-co-(e-caprolactone)],polycarbonates, poly(amino acids), poly(hydroxyalkanoate)s,polyanhydrides, polyorthoesters and blends of these polymers.

When described additives are included, they can be present in amounts,calculated on a dry weight basis, of from below 1 percent to as high as80 percent, based on total weight of the fibrous material. Moretypically, amounts range from between about 0.5 percent to about 50percent by weight, e.g., 5 percent, 10 percent, 20 percent, 30, percentor more, e.g., 40 percent.

Any additives described herein can be encapsulated, e.g., spray dried ormicroencapsulated, e.g., to protect the additives from heat or moistureduring handling.

The fibrous materials, densified fibrous materials, resins or additivescan be dyed. For example, the fibrous material can be dyed beforecombining with the resin and compounding to form composites. In someembodiments, this dyeing can be helpful in masking or hiding the fibrousmaterial, especially large agglomerations of the fibrous material, inmolded or extruded parts, when this is desired. Such largeagglomerations, when present in relatively high concentrations, can showup as speckles in the surfaces of the molded or extruded parts.

For example, the desired fibrous material can be dyed using an acid dye,direct dye or a reactive dye. Such dyes are available from Spectra Dyes,Kearny, N.J. or Keystone Aniline Corporation, Chicago, Ill. Specificexamples of dyes include SPECTRA™ LIGHT YELLOW 2G, SPECTRACID™ YELLOW4GL CONC 200, SPECTRANYL™ RHODAMINE 8, SPECTRANYL™ NEUTRAL RED B,SPECTRAMINE™ BENZOPERPURINE, SPECTRADIAZO™ BLACK OB, SPECTRAMINE™TURQUOISE G, and SPECTRAMINE™ GREY LVL 200%, each being available fromSpectra Dyes. In some embodiments, resin color concentrates containingpigments are blended with dyes. When such blends are then compoundedwith the desired fibrous material, the fibrous material can be dyedin-situ during the compounding. Color concentrates are available fromClariant.

It can be advantageous to add a scent or fragrance to the fibrousmaterials or densified fibrous materials.

Mobile Biomass Processing

Stationary processing facilities for processing biomass have beendescribed. However, depending upon the source of biomass feedstock andthe products produced therefrom, it can be advantageous to processbiomass in mobile facilities that can be located close to the source ofthe feedstock and/or close to target markets for products produced fromthe feedstock. As an example, in some embodiments, various grasses suchas switchgrass are used as biomass feedstock. Transporting large volumesof switchgrass from fields where it grows to processing facilitieshundreds or even thousands of miles away can be both wastefulenergetically and economically costly (for example, transportation offeedstock by train is estimated to cost between $3.00 and $6.00 per tonper 500 miles). Moreover, some of the products of processing switchgrassfeedstock can be suitable for markets in regions where biomass feedstockis grown (e.g., ruminant feed for livestock). Once again, transportingruminant feed hundreds or thousands of miles to market can not beeconomically viable.

Accordingly, in some embodiments, the processing systems disclosedherein are implemented as mobile, reconfigurable processing facilities.One embodiment of such a mobile facility is shown in FIG. 41. Processingfacility 8000 includes five transport trucks 8002, 8004, 8006, 8008, and8010 (although five trucks are shown in FIG. 41, in general, any numberof trucks can be used). Truck 8002 includes water supply and processingsystems and electrical supply systems for the other trucks. Trucks 8004,8006, 8008, and 8010 are each configured to process biomass feedstock inparallel.

Truck 8002 includes a water supply inlet 8012 for receiving water from acontinuous supply (such as a water main) or a reservoir (e.g., a tank onanother truck, or a tank or other reservoir located at the processingsite). Process water is circulated to each of trucks 8004, 8006, 8008,and 8010 through a water supply conduit 8020. Each of trucks 8004, 8006,8008, and 8010 includes a portion of conduit 8020. When the trucks arepositioned next to one another to set up the mobile processing facility,the portions of conduit 8020 are connected to form a continuous watertransport conduit. Each of trucks 8004, 8006, 8008, and 8010 includes awater inlet 8022 to supply process water, and a water outlet 8024 toremove used process water. The water outlets 8024 in each of trucks8004, 8006, 8008, and 8010 lead to a piecewise continuous water disposalconduit 8026, which is similarly joined into a continuous conduit whenthe trucks are positioned next to one another. Waste process water iscirculated to water processor 8028 in truck 8002, which treats the waterto remove harmful waste materials and then recycles the treated watervia conduit 8030 back into supply conduit 8020. Waste materials removedfrom the used process water can be disposed of on site, or stored (e.g.,in another truck, not shown) and transported to a storage facility.

Truck 8002 also includes an electrical supply station 8016 that provideselectrical power to each of trucks 8004, 8006, 8008, and 8010.Electrical supply station 8016 can be connected to an external powersource via connection 8014. Alternatively, or in addition, electricalsupply station can be configured to generate power (e.g., via combustionof a fuel source). Electrical power is supplied to each of trucks 8004,8006, 8008, and 8010 via electrical supply conduit 8040. Each of trucks8004, 8006, 8008, and 8010 includes an electrical power terminal 8018 towhich devices on the truck requiring electrical power are connected.

Each of trucks 8004, 8006, 8008, and 8010 includes a feedstock inlet8042 and a waste outlet 8044. Biomass feedstock enters each of trucks8004, 8006, 8008, and 8010 through inlet 8042, where it is processedaccording to the methods disclosed herein. Following processing, wastematerial is discharged through outlet 8044. Alternatively, in someembodiments, each of trucks 8004, 8006, 8008, and 8010 can be connectedto a common feedstock inlet (e.g., positioned in truck 8002), and eachtruck can discharge waste material through a common outlet (e.g., alsopositioned in truck 8002).

Each of trucks 8004, 8006, 8008, and 8010 can include various types ofprocessing units; for example, in the configuration shown in FIG. 41,each of trucks 8004, 8006, 8008, and 8010 includes an ion accelerator8032 (e.g., a horizontal Pelletron-based tandem folded accelerator), aheater/pyrolysis station 8034, a wet chemical processing unit 8036, anda biological processing unit 8038. In general, each of trucks 8004,8006, 8008, and 8010 can include any of the processing systems disclosedherein. In certain embodiments, each of trucks 8004, 8006, 8008, and8010 will include the same processing systems. In some embodiments,however, one or more trucks can have different processing systems.

In addition, some or all trucks can have certain processing systemsonboard but which are not used, depending upon the nature of thefeedstock. In general, the layout of the various onboard processingsystems on each of trucks 8004, 8006, 8008, and 8010 is reconfigurableaccording to the type of material that is processed.

Processing facility 8000 is an exemplary parallel processing facility;each of trucks 8004, 8006, 8008, and 8010 processes biomass feedstock inparallel. In certain embodiments, mobile processing facilities areimplemented as serial processing facilities. An embodiment oftrain-based serial mobile processing facility 8500 is shown in FIG. 42.Processing facility 8500 includes three rail cars 8502, 8504, and 8506(in general, any number of rail cars can be used), each configured toperform one or more processing steps in an overall biomass processingprocedure. Rail car 8502 includes a feedstock inlet for receivingfeedstock from a storage repository (e.g., a storage building, oranother rail car). Feedstock is conveyed from one processing unit toanother among the three rail cars via a continuous conveyor system. Railcar 8502 also includes an electrical supply station 8514 for supplyingelectrical power to each of rail cars 8502, 8504, and 8506.

Rail car 8502 includes a coarse mechanical processor 8516 and a finemechanical processor 8518 for converting raw feedstock to a finelydivided fibrous material. A third mechanical processor 8520 rolls thefibrous material into a flat, continuous mat. The mat of fibrousmaterial is then transported to an ion accelerator 8522 on rail car 8504that exposes the fibrous material to an ion beam. Following exposure tothe ion beam, the fibrous material is transported to a low energyelectron accelerator 8524.

The fibrous material is subsequently transported to a chemicalprocessing unit 8526 on rail car 8506 for one or more chemical treatmentsteps. Rail car 8506 includes a process water inlet 8532 which receivesprocess water from an external reservoir (e.g., a tank or another railcar).

Following chemical treatment in processing unit 8526, the material istransported to a biological processing unit 8528 to initiatefermentation of liberated sugars from the material. After biologicalprocessing is complete, the material is transported to a separator 8530,which diverts useful products into conduit 8510 and waste materials intoconduit 8512. Conduit 8510 can be connected to a storage unit (e.g., atanker car or an external storage tank). Similarly, waste products canbe conveyed through conduit 8512 to a storage unit such as a tanker car,and/or to an external storage facility. Separator 8530 also recyclesclean process water for subsequent delivery to chemical processing unit8536 and/or biological processing unit 8528.

As discussed previously, processing facility 8500 is an example of asequential configuration of a mobile processing facility; each of railcars 8502, 8504, and 8506 includes a different subset of processingsystems; and the feedstock process flow from each car is connected tothe next car in series to complete the processing sequence.

In general, a wide variety of different mobile processing configurationscan be used to process biomass feedstock. Both truck-based andtrain-based mobile processing facilities can be configured for eitherserial operation or parallel operation. Generally, the layout of thevarious processing units is reconfigurable, and not all processing unitscan be used for particular feedstocks. When a particular processing unitis not used for a certain feedstock, the processing unit can bewithdrawn from the process flow. Alternatively, the processing unit canremain in the overall process flow, but can be deactivated so thatfeedstock passes through the deactivated unit rapidly without beingmodified.

Mobile processing facilities can include one or more electronic controldevices that automate some or all aspects of the biomass processingprocedure and/or the mobile facility setup procedure. For example, anelectronic control device can be configured to receive input informationabout a feedstock material that is to be processed, and can generate avariety of output information including a suggested configuration of themobile processing facility, and/or values for one or more processparameters involved in the biomass processing procedure that will beimplemented.

While transportation by truck has been described above, part or all ofthe processing facility may be transported by any other means, forexample by rail or by a nautical vessel, e.g., a ship, barge, boat,dock, or floating platform. Transporting may also be performed usingmore than a single mode of transport, e.g., using a container on both aship and a tractor trailer or train.

In some embodiments, the methods described herein can be performedusing, for example, coal (e.g., lignite coal).

Accordingly, other embodiments are within the scope of the followingclaims.

What is claimed is:
 1. A product comprising recalcitrance reducedlignocellulosic biomass in densified form and an enzyme.
 2. The productof claim 1 wherein the lignocellulosic biomass comprises corn cobsand/or corn stover.
 3. The product of claim 1 wherein the enzymecomprises a cellulolytic enzyme.
 4. The product of claim 1 wherein theenzyme comprises a multiple enzyme cocktail.
 5. The product of claim 1wherein the recalcitrance reduced lignocellulosic biomass has beenpretreated with an electron beam.
 6. The product of claim 1 wherein theenzyme is present at a dosage of between 5.0 and 50 Filter Paper Units(FPU or IU) per gram of cellulose.
 7. The product of claim 1 wherein theenzyme comprises a dried enzyme.
 8. The product of claim 1 wherein theenzyme comprises an enzyme requiring activation.
 9. The product of claim1 wherein the enzyme is selected from the group consisting of phytase,cellulase, lactase, lipase, pepsin, catalase, xylanase, pectinase, andmixtures thereof.
 10. A method of making a feed material, the methodcomprising densifying a recalcitrance reduced lignocellulosic biomassand combining with an enzyme.
 11. The method of claim 10 furthercomprising, prior to densifying, preparing the recalcitrance reducedlignocellulosic biomass by treating a lignocellulosic biomass withelectron beam radiation.
 12. The method of claim 11 wherein treatingwith electron beam irradiation comprises applying a dosage of greaterthan 5 Mrad of radiation to the biomass.
 13. The method of claim 10wherein the lignocellulosic biomass comprises corn cobs and/or cornstover.
 14. The method of claim 10 wherein the enzyme comprises acellulolytic enzyme.
 15. The method of claim 1 wherein the enzymecomprises a multiple enzyme cocktail.
 16. The method of claim 10 whereindensifying comprises pelletizing.
 17. The method of claim 10 whereindensifying comprises applying a liquid to the recalcitrance reducedbiomass.
 18. The method of claim 10 wherein the enzyme comprises a driedenzyme.
 19. The method of claim 10 wherein the enzyme comprises anenzyme requiring activation.
 20. The method of claim 10 wherein theenzyme is selected from the group consisting of phytase, cellulase,lactase, lipase, pepsin, catalase, xylanase, pectinase, and mixturesthereof.